RU2554774C2 - Methods and reagents for improvement of detecting amyloid beta-peptides - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relate to medicine and deals with method of diagnosing neurodegenerative disease in individual, including the following stages (i) determination of one or several parameters, selected from group, consisting of 3ab40 or value of calculated parameter, selected from group, consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+1ab42+2ab42; (ii) comparison of parameter value with standard value, corresponding to value of said parameter in standard sample; and (iii) diagnostics of neurodegenerative disease, in case if increase of parameter value in comparison with standard value is observed. Group of inventions also deals with method of detecting stage, preceding neurodegenerative disease, method of differentiating neurodegenerative disease from stage, preceding said neurodegenerative disease.

EFFECT: group of inventions provide high sensitivity and specificity of detection methods.

13 cl, 12 ex, 14 dwg, 12 tbl

Description

FIELD OF THE INVENTION

The present invention relates to the field of immunoassays, and more particularly to methods for increasing the sensitivity of immunoassays for the determination of amyloid beta peptides in biological fluids.

State of the art

Alzheimer's disease (AD) is a progressive monetary disease of the central nervous system, characterized by progressive and increasing loss of memory, followed by impaired functioning of the motor functions of the limbs and the whole organism and, ultimately, death. This disease is obviously the most common cause of dementia, which is observed in 1-6% of the population aged 65 years and above and in 10-20% of the population aged over 80 years.

AD differs from other types of dementia in several pathological signs, including the progressive formation of senile plaques in the extracellular space between the neurons of the patient’s brain. Amyloid deposits are formed in the core of the plaques, formed mainly by peptide fibrils consisting of 40-42 amino acids and called the amyloid β-peptide (Αβ), which is surrounded by degenerated neurites and glial cells. Such a peptide is formed by proteolytic processing of a precursor protein called a β-amyloid precursor protein (βΑΡΡ).

AD can be classified in accordance with the age-related manifestation of this disease, that is, at an earlier age (up to 60 years) and at a later age (over 60 years old) and with autosomal dominant inheritance, that is, as hereditary or sporadic BA. The early manifesting hereditary forms of AD can be associated with a known mutation in the genes encoding βΑΡΡ, presenilin 1 and presenilin 2 (and located, respectively, on chromosomes 21, 14 and 1). These classifications are not mutually exclusive. The most common forms are sporadic forms, manifesting at a later age.

In clinical practice, the diagnosis of AD is made according to clinical criteria, based on the presence of typical clinical signs and the exclusion of other types of dementia using neuroimaging methods and blood tests. The reliability of the diagnosis based on these criteria is acceptable, although in accordance with studies conducted after autopsy of the brain, it was found that 10-20% of patients with diagnosed AD had another disease. In addition, modern diagnostic methods can only be implemented if the neurodegenerative process is already so advanced that the patient suffers from several forms of dementia, and the brain damage is so extensive that it limits the choice of therapeutic measures. For a final diagnosis, a pathological and anatomical study of brain tissue is necessary.

Taking into account the fact that Αβ accumulates in the brain of patients with AD and is a central element in the pathogenesis of AD, it should be noted that this protein is considered as the most suitable candidate for its use as an AD biomarker. However, the use of Αβ as a biomarker of AD present in plasma is associated with the problem that serum concentrations of Αβ (Αβ (1-40) and Αβ (1-42)) peptides are extremely low, and therefore do not yet exist analyzes that would have a sensitivity sufficient to ensure reliable detection of these peptide molecules.

Many different assays have been performed to determine the levels of amyloid beta peptides in biological samples (see, for example, the methods described by Scheuner et al. (Nature Med., 1996, 2: 864-870); Tamaoka A et al. (J. Neurol. Sci., 1996, 141, 65-68); Suzuki, N. et al. (Science, 1994, 264: 1336-1340); WO200722015, Vanderstichele H et al. (Amyloid, 2000, 7, 245-258 ); Fukomoto y col. (Arch. Neurol. 2003, 60, 958-964); Mehta et al. (Arch. Neurol. 57, 2000, 100-105); Mayeux, R. et al. (Ann Neurol. 1999 46, 412-416); Lanz, TA and Schacthter, JB (J. Neuroscience Methods, 2006, 157: 71-81), and WO200750359, WO0162801, WO0315617, WO0246237, WO0413172, however, all ELISA assays known in currently, have a low detection limit, in all likelihood not falling into the range of unambiguous pg / ml values, which is sufficient for detecting Αβ40 and Αβ42 in CSF, as well as for detecting these molecules in the plasma of patients suffering from hereditary AD, and are unsuitable for detecting Αβ42 in the plasma of patients suffering from sporadic AD, in which the concentration of β42 in plasma is much lower.

To date, analyzes performed on only one Αβ peptide have a detection limit below single digits in the pg / ml range, as described in WO200646644 and WO2009015696.

WO200646644 describes an electrochemiluminescent (ECL) sandwich assay where mAb 21F12 (recognizing amino acids 33-42 Α β42) is bound to magnetic spheres, which are then used to capture the Αβ42 peptide in a sample containing Αβ42 and contacted with a 3D6 mAb bound to with a ruthenium complex. Then, the amount of bound 3D6 antibody is detected by the luminescent radiation emitted by the ruthenium complex when electrical energy is communicated to it. The use of such an analysis allows the authors of the present invention to detect at least 0.5 pg / ml standard Αβ42. However, if the same analysis is used to compare Αβ42 in plasma samples in patients with AD and in healthy patients, then there can be no significant difference between patients of these two groups, as a result of which the authors of the present invention concluded that the amount of intact Αβ42 in serum is very low due to its degradation, and returned to competitive ELISA using mAb 21F12, which gives a lower level of sensitivity in the range of the order of ng / ml.

WO2009015696 describes a highly sensitive ELISA sandwich assay in which a detectable antibody is contacted with a biotin-labeled reagent specific for said antibody. This reagent is contacted with streptavidin bound to peroxidase. Then peroxidase activity is detected by the colorimetric method using TMB reagent or the fluorescence method using QuantaBlue.

WO2006053251 describes a method for determining amyloid beta peptide molecules in a sample, where the method involves contacting the sample with a denaturing agent, extracting the peptide pool from a sample denaturing agent mixture, separating the amyloid beta peptide molecules from the specified pool, and determining the number of amyloid beta molecules peptides. This method requires the stage of separation of peptides before they are determined, which increases the time it takes to implement this method and the cost of its implementation.

Therefore, it is necessary to develop improved immunological assays and obtain kits for detecting peptides derived from Αβ, which will solve the problems associated with the use of known methods and kits, and in particular, to develop assays and kits with a sensitivity sufficient for reliable detection of Αβ peptides in the plasma of patients suffering from sporadic asthma.

Description of the invention

In its first aspect, the present invention relates to a method for diagnosing a neurodegenerative disease in an individual, detecting a condition preceding a neurodegenerative disease, or differentiating a neurodegenerative disease from a condition preceding said neurodegenerative disease, wherein said method comprises the steps of:

(i) determining one or more parameters selected from the group consisting of:

(a) the level of one or more free amyloid beta peptides in a biological sample of said individual;

(b) aggregate levels of one or more free amyloid peptides in a biological sample of said individual and said one or more amyloid beta peptides associated with macromolecular components present in said biological sample, where said levels of aggregates are determined by assessing the amount of said one or more amyloid beta-peptides in the cell-free fraction of the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for promoting dissociation of amyloid beta peptide or amyloid beta peptides from components present in a biological sample,

(c) the level of one or more amyloid beta peptides associated with cells in a biological sample of a specified individual, where the specified level is determined by isolating the cell fraction of the specified biological sample, contacting the specified cell fraction of the specified sample with a protein-solubilizing agent under conditions suitable for stimulation dissociation of amyloid beta peptide or amyloid beta peptides from cells present in the specified sample;

(ii) comparing the value of at least one of the parameters (b) or (c), or the value of a parameter calculated by arithmetically combining at least two parameters (a) to (c) with a reference value corresponding to the value of said parameters (b) or (c) or a specified calculated parameter in a reference sample; and

(iii) diagnosing a neurodegenerative disease; detecting a condition preceding a neurodegenerative disease; or differentiation of a neurodegenerative disease from a condition preceding said neurodegenerative disease, if there is a change in the value of the parameter or the value of the calculated parameter compared to the reference value.

In its second aspect, the present invention relates to a kit for determining amyloid beta peptides in a biological sample, where the specified kit includes:

(i) a protein solubilizing agent and

(ii) at least an anti-amyloid beta peptide antibody.

A brief description of the graphic material

Figure 1: A-F. Dotted measurement curves for (A) UP Αβ1-40, (B) DP Αβ1-40, (C) CBAβ1-40, (D) UP Αβ1-42, (E) DP Αβ1-42 and (F) CB Αβ1 -42 obtained in two independent laboratories (Lab1 and Lab2). Most points are close to the concordance line, which indicates a significant and almost accurate degree of conformity of measurements. On each graph, the inset below the right shows the correlation and concordance coefficient (CCC) and 95% confidence intervals.

Figure 2: Direct markers Αβ1-40 (A), Αβ1-42 (B) and calculated markers Αβ1-40 and Αβ1-42 (C). A. Concentrations (pg / ml) of free Αβ1-40 in serum (FP), levels of total Αβ1-40 in plasma (including free Αβ1-40 and Αβ1-40 associated with plasma components) (TP) and Αβ1-40 associated with cells (CB) in healthy control individuals (HC), in patients with mild cognitive impairment (MCI), and in patients with Alzheimer's disease (AD). B. Concentrations (pg / ml) of free Αβ1-42 in serum (FP), total plasma levels of Αβ1-42 (including free Αβ1-42 and Αβ1-42 associated with plasma components) (TP) and Αβ1-42 bound with cells (CB) in healthy control individuals (HC), in patients with mild cognitive impairment (MCI), and in patients with Alzheimer's disease (AD). C. Values, pg / ml, for aggregated total Αβ1-40 in plasma (obtained by contacting a plasma sample with a protein-solubilizing agent) plus cell-bound Αβ1-40 (TP + CB Αβ1-40), total Αβ1-42 in plasma (obtained upon contact of a plasma sample with a protein-solubilizing agent) plus Αβ1-42 (TP + CB Αβ1-42) bound to cells, or the values of aggregated TP + CB Αβ1-40 and Αβ1-42 (TP + CB Αβ1-42). H, M, and A mean significant values (p <0.05) for healthy control (ZK) individuals, for patients with mild cognitive impairment (MCI), and for patients with AD, respectively. * means p <0.01.

Figure 3: A-F. The curves of the values of (A) DP Αβ1-40, (B) CB Αβ1-40, (C) UP Αβ1-42, (D) DP Αβ1-42, (E) T40 and (F) Τ-βΑΡΒ for SC individuals, patients with MCI and patients with AD. The numbers co * mean the values obtained for individual patients in the MCI and BA groups, which, for accuracy, are not given on the same scale on the ordinate axis. The horizontal line represents the boundary values obtained for patients with MCI and ZK individuals.

Figure 4: ROC curve for the marker lab40 in patients with MCI / HC. Area under the ROC curve = 0.510.

Figure 5: ROC curve for marker 2ab40 in patients with MCI / HC. Area under the ROC curve = 0.778.

Figure 6: ROC curve for marker 3ab40 in patients with MCI / HC. Area under the ROC curve = 0.458.

Figure 7: ROC curve for the marker lab42 in patients with MCI / HC. Area under the ROC curve = 0.576.

Figure 8: ROC curve for marker 2ab42 in patients with MCI / HC. Area under the ROC curve = 0.667.

Figure 9: ROC curve for marker 3ab42 in patients with MCI / HC. Area under the ROC curve = 0.744.

Figure 10: ROC curve for marker 3ab42 in patients with MCI / HC. Area under the ROC curve = 0.508.

Figure 11: ROC curve for 2ab40 + 3ab40 markers in patients with MCI / HC. Area under the ROC curve = 0.830.

Figure 12: ROC curve for 2ab42 + 3ab42 markers in patients with MCI / HC. Area under the ROC curve = 0.713.

Figure 13: ROC curve for 2ab42 + 3ab42 markers in patients with MCI / HC. Area under the ROC curve = 0.777.

Figure 14: ROC curve for markers 2ab40 + 3ab40 + 2ab42 + 3ab42 in patients with MCI / HC. Area under the ROC curve = 0.848.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have unexpectedly found that dilution of plasma with sample buffer leads to an increase in detectable levels of Αβ1-40 and Αβ1-42. Not limited to any theory, it can be noted that such a plasma dilution leads to a change in the ionic strength and molecular interactions with the sample, followed by the release of Αβ1-40 and Αβ1-42 associated with proteins and other plasma components.

Thus, the increase in the values measured after plasma dilution may be due to the detection of Αβ peptides released from proteins and other plasma components, and can be interpreted as the value of the total plasma Αβ level.

In any case, these results show that the levels of the Αβ peptide in the blood are much higher than the levels that could be obtained by simple analysis in undiluted plasma. A complete determination of total blood βΑΡΒ levels should include a quantitative assessment of plasma free peptides, peptides bound to plasma proteins, and peptides bound to blood cells. Such an exhaustive quantitative assessment of the various components of βΑΡΒ will make it possible to more accurately measure the levels of β in the blood and makes it possible to better understand the complex mechanism of regulation of β peptides in a healthy and sick person. In addition, the levels of these pools of amyloid beta peptides, as well as the value of some calculated parameters obtained by arithmetically adding the concentrations of different pools and concentrations of free amyloid beta peptides, can be used to determine if a patient is suffering from a neurodegenerative disease or condition preceding a neurodegenerative disease, and in order to differentiate a neurodegenerative disease from a condition preceding said neurodegenerate a new disease.

Diagnostic method according to the invention

Thus, in its first aspect, the present invention relates to a method for diagnosing a neurodegenerative disease in an individual, detecting a condition preceding a neurodegenerative disease, or differentiating a neurodegenerative disease from a condition preceding said neurodegenerative disease, wherein said method comprises the steps of:

(i) determining one or more parameters selected from the group consisting of:

(a) the level of one or more free amyloid beta peptides in a biological sample of said individual;

(b) aggregate levels of one or more free amyloid peptides in a biological sample of said individual and said one or more amyloid beta peptides associated with macromolecular components present in a biological sample, wherein said aggregate levels are determined by evaluating the amount of said one or more amyloid beta -peptides in the cell-free fraction of the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable to stimulate the dissociation of the amyloid beta-peptide or amyloid beta-peptides of the components present in a biological sample;

(c) the level of one or more amyloid beta peptides associated with cells in a biological sample of a specified individual, where the specified level is determined by isolating the cell fraction of the specified biological sample, contacting the specified cell fraction of the specified sample with a protein-solubilizing agent under conditions suitable for stimulation dissociation of amyloid beta peptide or amyloid beta peptides from cells present in the specified sample; and

(ii) comparing the value of at least one of the parameters (b) or (c), or the value of the parameter calculated by arithmetically combining at least two parameters (a) to (c), with a reference value corresponding to the value of the specified parameters (b ) or (c), or the specified calculated parameter in the reference sample; and

(iii) diagnosing a neurodegenerative disease, detecting a condition preceding a neurodegenerative disease, or differentiating a neurodegenerative disease from a condition preceding said neurodegenerative disease, if there is a change in the value of the parameter or the value of the calculated parameter compared to the reference value.

As used herein, the term “diagnosis” includes evaluating an individual’s predisposition to develop a disease, determining the presence of a disease in an individual, and predicting the outcome of a disease in an individual suffering from the disease. As is known to those skilled in the art, establishing such a diagnosis in an individual usually cannot be 100% accurate, although it is preferred that it is accurate. However, by this term is meant that a statistically significant group of individuals can be identified as individuals suffering from or having a predisposition to the disease. If this group is statistically significant, then it can be easily determined by a specialist using several well-known methods of statistical estimation, for example, by determining confidence intervals and p-values, applying the Student t-test, Mann-Whitney criterion, etc. Detailed descriptions are given in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p values are preferably 0.2, 0.1 or 0.05.

As used herein, the term "individual" refers to all animals classified as mammals, including, but not limited to, domestic and farm animals, primates and humans, for example humans, primates, with the exception of humans, cows, horses, pigs, sheep, goats, dogs, cats or rodents. The preferred individual is a person, that is, a man or woman of any age or race.

As used herein, the term "neurodegenerative disease" means a condition or disorder in which loss of neuronal cells occurs as a result of their death, which leads to a deterioration in cognitive function or to damage or dysfunction of the nervous system, or to complications that may be characterized as neurological, neurodegenerative, physiological, mental or behavioral disorders. Suitable neurodegenerative diseases that can be diagnosed by the methods of the invention include, but are not limited to, age-related macular degeneration; Creutzfeldt-Jakob disease; Alzheimer's disease; radiation therapy dementia; axon damage; acute disseminated depression caused by dysfunction of the cerebral cortex, alpha synucleinopathy, cerebral ischemia, Huntington's disease, permanent focal cerebral ischemia of the cerebral cortex, peripheral nerve regeneration, post-epileptic status model, spinal cord injury, sporadic amyotrophic lateral sclerosis and transmissive encephalitis.

In a preferred embodiment of the invention, the neurodegenerative disease is Alzheimer's disease. The term "Alzheimer's disease" (or "senile dementia") means a mental illness associated with a specific degenerative brain disease, which is characterized by the presence of senile plaques, neuritic tangles and progressive loss of neurons and is clinically manifested by progressive memory loss, confusion, behavioral disorders, disability , a gradual deterioration of physical condition and ultimately leads to death. Patients with Alzheimer's disease were identified using the NINCDS-ADRDA criterion (CDR = 1, MMSE from 16 to 24 points and atrophy of the medial portion of the temporal region (as determined by MRI)> 3 points according to the Shelten scale).

As used herein, the term "stage preceding a neurodegenerative disease" means a condition that can be defined as a transition from a condition observed in a normal individual to a condition defined as a neurodegenerative disorder, and which is characterized by the presence of some signs and symptoms of a neurodegenerative disorder or a subgroup of signs and symptoms observed in patients suffering from neurodegenerative disorder. In a preferred embodiment of the invention, the stage preceding said neurodegenerative disease is a mild cognitive impairment (hereinafter referred to as MCI), which means a transitional stage from cognitive impairment in natural aging to early Alzheimer's disease. Patients are usually identified as patients with MCI if they meet the Mayo clinical criterion (CDR = 0.5, which indicates atrophy of the medial portion of the temporal region (determined by MRI), and this score is 3 points higher than the Shelten score, which indicates a picture of hypometabolism in the parietal and / or temporal region, determined by positron emission tomography performed using 18-fluorodeoxyglucose (PET-FDG)) (presumably AD).

The term "differentiation of a neurodegenerative disease from the stage preceding a neurodegenerative disease" means the ability to distinguish a patient having a condition preceding a neurodegenerative disease or a condition in the prodromal period of the disease from a patient already suffering from this disease. In the specific case where the neurodegenerative disease is Alzheimer's disease, the method according to the invention allows to differentiate Alzheimer's disease from the prodromal stage of the disease, known as mild cognitive impairment (MCI).

In its first stage, the method according to the invention includes determining at least one parameter selected from the group consisting of:

(a) the level of one or more free amyloid beta peptides in a biological sample of said individual;

(b) aggregate levels of one or more free amyloid peptides in a biological sample of said individual and said one or more amyloid beta peptides associated with macromolecular components present in said biological sample, where said levels of aggregates are determined by assessing the amount of said one or more amyloid beta-peptides in the cell-free fraction of the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptide or amyloid beta peptides from components present in a biological sample;

(c) the level of one or more amyloid beta peptides associated with cells in a biological sample of a specified individual, where the specified level is determined by isolating the cell fraction of the specified biological sample, contacting the specified cell fraction of the specified sample with a protein-solubilizing agent under conditions suitable for stimulation dissociation of amyloid beta peptide or amyloid beta peptides from cells present in the specified sample.

As used herein, the term "amyloid beta peptide" is synonymous with the terms "A-beta", "Abeta", "beta-AP", "A-beta-peptide" or "Αβ-peptide" and means a family of peptides that are basic chemical components of senile plaques and vascular amyloid deposits (amyloid angiopathy) present in the brain of patients with Alzheimer's disease (AD), patients with Down syndrome and patients suffering from hereditary cerebral hemorrhage associated with Dutch type amyloidosis (HCHWA-D). Amyloid beta peptides are fragments of an amyloid beta precursor protein (APP) that contains a different number of amino acids, usually 38-43 amino acids.

Amyloid beta peptides are usually referred to as “Αβ (x-y),” where x is the number of amino acids at the amino end of the amyloid beta peptide and y is the number of amino acids at the carboxy end. For example, Αβ (1-40) is an amyloid beta peptide whose amino end begins at position 1 of the amino acid sequence, and the carboxy end ends at position 40 of the amino acid sequence, where this sequence is represented by SEQ ID NO: l. Examples of amyloid beta peptides that can be determined by the method according to the invention are, but are not limited to, Αβ (1-38) (SEQ ID NO: 2), Αβ (1-39) (SEQ ID NO: 3), Αβ (1-40) (SEQ ID ΝΟ: 1), Αβ (1-41) (SEQ ID NO: 4), Αβ (1-42) (SEQ ID NO: 5), Αβ (1-43) (SEQ ID NO: 6), Αβ (11-42) (SEQ ID NO: 7), Αβ (3-40) (SEQ ID NO: 8), Αβ (3-42) (SEQ ID NO: 9), Αβ (4 -42) (SEQ ID NO: 10), Αβ (6-42) (SEQ ID NO: 11), Αβ (7-42) (SEQ ID NO: 12), A13 (8-42) (SEQ ID NO: 13), Αβ (9-42) (SEQ ID NO: 14), Αβ (x-40), Αβ (x-42) and Αβ (x-38), as well as all amyloid beta peptides that belong to the set species of amyloid beta peptides, where individual species are unidentified. In preferred embodiments of the invention, amyloid beta peptides that can be detected by the method of the invention are Αβ (1-40) and Αβ (1-42).

As used herein, the term “Αβ (1-42)” means a peptide that consists of 42 amino acids corresponding to amino acids 672-713 (SEQ ID NO: 5) APP, and which is obtained by sequential proteolytic cleavage of the amyloid precursor protein (SEQ ID NO: 15) under the influence of β- and γ-secretases.

As used herein, the term “Αβ (1-40)” means a peptide that consists of 40 amino acids corresponding to amino acids 672-711 (SEQ ID NO: 1), and which is obtained by sequential proteolytic cleavage of the amyloid precursor protein (SEQ ID NO: 15 ) under the action of β- and γ-secretases.

As used herein, the term “biological sample” includes (1) body fluids such as whole blood, serum, plasma, urine, lymph, saliva, sperm, sputum, tears, mucus, sweat, milk, brain extracts, and cerebrospinal fluid; (2) blood components such as plasma, serum, blood cells, and platelets; (3) extracts obtained from hard tissues or organs such as the brain; and (4) extracts obtained from cultures of cell lines or primary cells of a human or animal, such as primary human neurons and primary neurons isolated from transgenic mice carrying human APP genes, e.g. PDAPP cells isolated from a transgenic animal (e.g., mouse) as well as human kidney cell line 293, human neuroglioma cell line, human HeLa cell line, primary endothelial cell line (e.g., HUVEC cells), primary human fibroblast line or primary lymphoblast line (including e Dogen cells derived from patients with APP-mutations), primary mixed cell culture of human brain (including neurons, astrocytes and glial cells) or cell line of Chinese hamster ovary (CHO). The methods of the invention are particularly suitable for measuring Αβ in a blood sample of a human or non-human animal, such as whole blood, plasma, or a sample containing any blood components in any quantity.

The term “whole blood” means blood taken from a human or animal and containing cellular components and a liquid component. Whole blood can be stored in a clotted state or in a non-clotted state. The term “whole blood” also includes blood in which a portion of the cellular components or all cellular components, such as white blood cells or red blood cells, have been lysed.

The term "plasma" means the liquid component of whole blood. Depending on the separation method used, plasma components may be completely absent in the plasma, or the plasma may contain a different number of platelets and / or a small amount of other cellular components.

The term "serum" means a plasma in which there is no coagulation factor, such as fibrinogen protein, and other blood coagulation factors.

As used herein, the term “free amyloid beta peptide” means amyloid beta peptides that are not associated with any component of a biological sample and which are readily available for binding to a specific antibody. This peptide can be determined by standard immunological methods by contacting a biological sample with an antibody specific for the specified peptide. In a preferred embodiment of the invention, the level of free amyloid peptide is determined in plasma.

As used herein, the term “amyloid beta peptide associated with macromolecular components” means an amyloid beta peptide that is non-covalently attached to molecules or attached to molecules present in a test biological sample. This peptide is usually difficult to access for immunological detection, and therefore, pre-treatment of such a biological sample is required to separate this peptide from its components. Under these conditions, the amyloid beta peptide associated with the macromolecular components will be released from these components and will be available for immunological detection with specific antibodies. Since the biological sample already contains a certain amount of free amyloid beta peptide, the total amount of free amyloid peptide after contacting the sample with a protein solubilizing agent will include a certain level of aggregates of such a commonly present free amyloid beta peptide and amyloid beta peptide released after protein treatment solubilizing agent. If it is necessary to determine the level of amyloid beta peptide associated with macromolecular components in a biological sample, this can be done by determining the level of free amyloid beta peptide before treatment with a protein-solubilizing agent and the level of free amyloid beta peptide after treatment with protein solubilizing agent and subtracting the first value from the second value. In order to implement the present invention, the level of aggregated free amyloid beta peptides is usually determined, which includes the level of commonly present free amyloid beta peptides, as well as the level of amyloid beta peptides released from macromolecular components after treatment with a protein solubilizing agent. Therefore, the parameter that is usually determined when a sample is processed to dissociate an amyloid peptide from macromolecular components is the sum of the amount of free peptide present in the sample and the peptide associated with the macromolecular components.

Macromolecular components of a sample that can bind to amyloid beta peptides and which can constitute a pool of amyloid beta peptides associated with macromolecular components include both proteins as well as peptides. In the specific case of the implementation of this method on blood or plasma samples, macromolecular components are, but are not limited to, blood proteins and lipids. Representative blood proteins are albumin, immunoglobulin G, immunoglobulin E, immunoglobulin M, immunoglobulin A, fibrinogen (fibrin and their decomposition products), antitrypsin alpha-1, prealbumin, acid glycoprotein alpha-1, fetoprotein alpha-1, gap macroglobulin, ceruloplasmin, transferrin, C3 / C4-beta-2-microglobulin, beta-lipoprotein, alpha, beta and gamma globulins, C-reactive protein (CRP), prothrombin, thyroxin-binding protein, transthyretin, etc. . Representative blood lipids are free fatty acids, cholesterol, triglycerides, phospholipids, sphingolipids, and the like. The amount of amyloid beta peptide associated with the macromolecular components can be determined by contacting the cell-free biological sample with a protein-solubilizing agent under conditions suitable for inducing the release of these amyloid beta peptides from the macromolecular components.

In the present description, the term "contacting" means adding to this sample a sufficient amount of a solution containing a protein-solubilizing agent, such that the concentration of the protein-solubilizing agent in the resulting mixture is sufficient to effectively solubilize the amyloid beta peptide that binds to proteins and cells in this sample. In this case, it is preferable that said protein-solubilizing agent is present in the buffer solution in an amount that would not cause a significant change in the pH of the sample.

As used herein, the term “protein solubilizing agent” means any compound of a particular composition capable of altering the secondary, tertiary and / or quaternary structure of polypeptides while maintaining their primary structure. As for the properties of these protein-solubilizing agents, these agents have the ability to increase the solubility of proteins in this sample, as well as prevent intermolecular and intramolecular protein aggregation. Protein solubilizing agents suitable for use in the present invention include, but are not limited to, detergents, chaotropic agents, reducing agents, or mixtures thereof.

The term “detergent” as used herein is generally synonymous with the term “surfactant” and means amphipathic surfactants which, when added to a liquid, reduce the surface tension in the liquid compared to the surface tension of the same liquid, in which this detergent is absent. Detergents can also prevent protein aggregation and non-specific interaction or binding with impurities that contaminate the protein of interest. Detergents suitable for use in the present invention include, but are not limited to, nonionic (neutral), anionic, cationic or zwitterionic detergents.

Examples of non-ionic or neutral detergents include, but are not limited to, Tween series detergents such as tween® 20, tween® 21, tween® 40, tween® 60, tween® 61, tween® 65, tween® 80, tween® 81, tween® 85, Span® series detergents such as Span® 20; detergents of a series of tergitols, such as tergitol type 15-S-12; Brij® series detergents such as Brij® 35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P; tween series detergents such as tween® 20, tween® 21, tween® 40, tween® 60, tween® 61, tween® 65, tween® 80, tween® 81, tween® 85; Triton® series detergents such as Triton® X-100, Triton® X-114, Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16, Triton® GR-5M, Triton ® X-102, Triton® X-l5, Triton® X-151, Triton® X-207, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X -705-70 or non-ionic conservative variant of at least one of these detergents.

Examples of anionic detergents include, but are not limited to, cholic acid and its derivatives, taurocholic acid, triton X-200, triton W-30, triton-30, triton-770, dioctyl sulfosuccinate, N-oxide N ₅ N-dimethyldodecylamine, 1- sodium alkyl sulfonates, N-lauroyl sarcosine or fatty acid salts.

Examples of cationic detergents include, but are not limited to, fatty acid mono- and dimethylamines, alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylamine acetates, trialkylammonium acetates, alkyldimethylbenzylammonium salts, alkylalkylidene-alkylpyridylalkylidene-alkylpyridyl-alkylpyridinylidene alkylamidomethylpyridinium salts, alkylquinolinium salts, alkyl isoquinolinium salts, N, N-alkylmethylpyrrolidinium salts, 1,1-dialkylpiperidinium salts, 4,4-dialkylthiamorpholinium salts, s whether 4,4-dialkylthiamorpholinium 1-oxide, methyl bis (alkyl ethyl) -2-alkylimidazolinium methyl sulfate (and other salts), methylbis (alkylamidoethyl) -2-hydroxyethylammonium methyl sulfate (and other salts), alkylamidopropyl-dimethylbenzylammonium salts, carboxyalkyl salts alkyldimethylammonium, alkylamine oxides, alkyldimethylamine oxides, poly (vinylmethylpyridinium) salts, poly (vinylpyridine) salts, polyethyleneimines, trialkylphosphonium bicarbonates (and other salts), trialkylmethylphosphonium salts, alkylethylmethylsulfonium salts and alkyldonium salts.

Examples of zwitterionic detergents include, but are not limited to, 3 - [(3-cholamidopropyl) dimethylammonia] -1-propanesulfonate (CHAPS); 3 - [(3-cholamidopropyl) dimethylammonia] -2-hydroxy-1-propanesulfonate (CHAPSO); N- (alkyl-C10-C16) -N, N-dimethylglycine-betaine (EMPIGEN BB); caprylylsulfobetaine (SB3-10); 3- [N, N-dimethyl (3-myristoylaminopropyl) ammonia] propanesulfonate (amidosulfobetaine-14; ASB-14); N-tetradecyl-N, N-dimethyl-3-ammonia-1-propanesulfonate (3-14-detergent; ZWITTERGENT); N-dodecyl-N, N'-dimethyl-3-ammonia-1-propanesulfonate; N-octadecyl-N, N-dimethyl-3-ammonia-1-propanesulfonate; N-decyl-N, N-dimethyl-3-ammonium-1-propanesulfonate; miratain CB; mirataine BB; mirataine CBR; miratain ACS; miracar 2MHT and miracar 2MCA.

In a preferred embodiment of the invention, the protein solubilizing reagent is a detergent. In an even more preferred embodiment, said detergent is Tween 20. In an even more preferred embodiment, Tween 20 is used at a concentration of 0.5%.

As used herein, the term “chaotropic agent” means a compound or mixture of compounds that cleave hydrogen bonds and disrupt hydrophobic interactions between and within proteins. Chaotropic agents used in high concentrations destroy the secondary structure of the protein and form proteins in solution that are themselves not soluble. Suitable chaotropic agents include, but are not limited to, urea, guanidinium isothiocyanate, sodium thiocyanate (NaSCN), guanidine-HCl, guanidinium chloride, guanidinium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, potassium tritium chloride or rubidium acetate.

As used herein, the term “reducing agent” means any compound or substance that retains sulfhydryl groups in a reduced state and restores intramolecular or intermolecular disulfide bonds. Thus, for example, reducing agents suitable for use in the method according to the invention are sulfhydryl or phosphine reducing agents. Examples of sulfhydryl reducing agents are dithiothreitol (DTT), dithioerythritol (DTE) and β-mercaptoethanol. Examples of phosphine reducing agents are tributylphosphine (TBP) and tris-carboxyethylphosphine (TCEP).

Typically, a biological sample is first processed to remove the cell fraction. The cell-free sample is then contacted with a protein-solubilizing agent. In a preferred embodiment of the invention, the sample is diluted with a buffer containing a protein-solubilizing agent. Typically, the sample is diluted 5-fold in a buffer solution containing Tween 20.

As used herein, the term “buffer solution” means any substance or mixture of compounds in a solution that have the ability to neutralize acids and bases, but which does not significantly affect the initial acidity or basicity of the solution. Suitable buffer solutions used in the method according to the invention include, but are not limited to, Tris buffer solution, phosphate buffer solution, borate buffer solution, carbonate buffer solution, sodium glycine hydroxide buffer solution, or the like. A preferred buffer solution is a phosphate buffer solution, such as phosphate buffered saline or PBS.

The amount of a solution containing a protein-solubilizing agent that is added to the biological sample is not critical, provided that a sufficient level of dissociation of the amyloid beta peptide is achieved. So, for example, biological fluid can be diluted in a solution containing a protein-solubilizing agent at a dilution of at least 1/2 (vol./about.), 1/3 (vol./about.), 1/4 (vol. v / v), 1/5 (v / v), 1/6 (v / v), 1/7 (v / v), 1/8 (v / v), 1 / 9 (v / v), 1/10 (v / v), 1/20 (v / v), 1/50 (v / v), 1/60 (v / v vol.), 1/80 (vol./vol.), 1/90 (vol./vol.), 1/100 (vol./vol.) or more. Specialists know that any combination of these protein-solubilizing agents to the indicated dilution degree can be used, provided that the final concentration of the protein-solubilizing agent is adequate to achieve the desired effect. So, for example, a solution containing a protein-solubilizing agent may include the specified (e) selected (e) protein-solubilizing (e) agent (s) in a concentration of from 0.001% to 0.5% (w / v). After dilution in said solution containing a protein-solubilizing agent, said biological fluid usually contains said surfactant (s) in a concentration of less than 0.1% (w / v), preferably less more than 0.6% (w / v), more preferably no more than 0.5% (w / v), most preferably no more than 0.45% (w / v) and still more preferably 0.5%.

Buffer systems suitable for use in the present invention are Tris-HCl buffers, including a salt such as NaCl or KCl, and optionally BSA. Specific buffer systems include, but are not limited to:

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA, 0.05% Tween-20, 1M GuHCl;

50 mM Tris-HCl, pH 8, 0.5 M KCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 0.5 M KCl, 0.05%; BSA, 0.05% Tween-20, 1M GuHCl;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA, 0.05% tween-80;

50 mM Tris-HCl, pH 8, 0.5 M KCl, 0.05%; BSA, 0.05% tween-80;

50 mM Tris-HCl, pH 8, 0.5M NaCl; 0.05%; BSA, 0.05% Triton X-100

50 mM Tris-HCl, pH 8, 0.5 M KCl, 0.05%; BSA, 0.05% Triton X-100;

50 mM Tris-HCl, pH 8; 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.1%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA, 0.1% Tween-20;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.1%; BSA, 0.1% Tween-20;

50 mM Tris-HCl, pH 8, 1M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 1.5 M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 2M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 2.5 M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 3M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA, 0.05% Tween-20, 10% DMSO;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA, 0.05% Tween-20, 0.5 M GuHCl;

50 mM Tris-HCl, pH 6, 0.5 M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 7, 0.5 M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 9, 0.5 M NaCl, 0.05%; BSA, 0.05% tween-20;

50 mM Tris-HCl, pH 8, 0.5 M NaCl, 0.05%; BSA

So, for example, if Tween 20 is used as a protein-solubilizing agent, then preferably its concentration is 0.004-0.02%, and more preferably 0.005-0.01% (w / v).

The contacting step is preferably carried out at a low temperature to inhibit the activity of proteolytic enzymes present in the sample. Suitable temperatures are temperatures of about 0-10 ° C, preferably about 3-5 ° C, for example, about 4 ° C.

After contacting the biological fluid with a solution containing a protein-solubilizing agent, both fluids can be mixed. Mixing can be carried out by mixing, preferably shaking, more preferably high speed shaking, and most preferably swirling, for at least 5 seconds, preferably at least 10 seconds, and more preferably at least for 15 seconds (e.g. 15-50 seconds). The preferred speed of said mixing, stirring, shaking, high speed shaking or swirling is at least 250 rpm, preferably at least 500 rpm, more preferably at least 1000 rpm, and most preferably about 2000 -2500 rpm.

The contacting step is carried out under conditions suitable to achieve partial or, preferably, complete dissociation of the amyloid beta peptide from the protein and lipids present in the biological sample. Such conditions can be adequately determined by one of ordinary skill in the art by monitoring the amount of amyloid beta peptide that can be detected prior to the contacting step, and which can gradually increase at different times after the contacting step. The time of the experiment can be determined as described in the example presented in the experimental part.

A person skilled in the art knows that if the level of amyloid beta peptide is determined by diluting a biological sample with a buffer containing a protein-solubilizing agent, then the level of free amyloid beta peptide determined by the immunological method is adjusted taking into account the coefficient used before dilution of the biological sample.

Thus, in a preferred embodiment of the invention, the parameter determined in step (i) of the method according to the invention is one or more parameters selected from the group consisting of the level of the free ABETA40 peptide in a biological sample taken from the indicated individual (hereinafter referred to as 1ab40 or UP Αβ (1-40)), the level of aggregates of the free ABETA40 peptide in the biological sample, and the ABETA40 peptide associated with the components of the specified biological sample obtained as described above (hereinafter referred to as 2ab40 or DP Αβ (1-40)), level the free ABETA42 peptide in the biological sample (hereinafter referred to as 1ab42 or UP Αβ (1-42)), and the levels of aggregates of the free ABETA42 peptide in the biological sample, and the ABETA42 peptide associated with the components of the biological sample obtained as described above (hereinafter referred to as 2ab42 or DP Αβ (1-42)).

As used herein, the term “cell-associated amyloid beta peptide” means an amyloid beta peptide that is non-covalently attached to the surface of cells present in a biological sample and which is unable to bind to antibodies added to the sample, and therefore is not immunologically detectable. Typically, if the biological sample is blood, then the amyloid beta peptide is associated with red blood cells, white blood cells, including neutrophils, eosinophils, basophils, lymphocytes and monocytes, and platelets.

The amount of amyloid beta peptide associated with the cells in a given sample can be determined, and this value can be used alone or in combination with other parameters related to the amyloid beta peptides used in the methods of the invention. For this purpose, it is first necessary to isolate the cell fraction from the biological sample. This can be done using any method known to those skilled in the art, such as centrifugation, sedimentation, filtration, and the like. After isolation of the cell fraction from the biological sample, the cells are contacted with a protein solubilizing agent.

Suitable protein solubilizing agents are detergents, chaotropic agents and reducing agents, as defined above, and commonly used in a buffer solution at the appropriate concentration. Suitable agents, buffer solutions and concentrations of agents in the buffer solution are described above. The contacting step is carried out mainly as described above in the method for releasing an amyloid peptide bound to components (proteins and lipids) of a biological sample. In a preferred embodiment of the invention, said protein solubilizing agent is a detergent. In an even more preferred embodiment of the invention, said detergent is Tween 20. Suitable concentrations of Tween 20, used as a protein solubilizing agent, are defined above and are 0.004-0.02%, and more preferably 0.005-0.01% (wt. /about.).

The contacting step is preferably carried out at a low temperature to inhibit the activity of proteolytic enzymes present in the sample. Suitable temperatures are about 0-10 ° C, preferably about 3-5 ° C, for example about 4 ° C.

Typically, the contacting step is carried out by resuspending the cell fraction in a biological sample with a solution containing a protein solubilizing agent. Said resuspension can be carried out by carefully pipetting by collection and release, by stirring, preferably shaking, more preferably high-speed shaking, and most preferably, vortex shaking, for at least 5 seconds, preferably at least 10 seconds and more preferably at least 15 seconds (e.g., 15-50 seconds). The preferred speed of said mixing, stirring, shaking, high speed shaking or swirling is at least 250 rpm, preferably at least 500 rpm, more preferably at least 1000 rpm, and most preferably about 2000 -2500 rpm.

The contacting step is carried out under conditions suitable to achieve partial or, preferably, complete dissociation of the amyloid beta peptide from cells present in the biological sample. Such conditions can be adequately determined by one of ordinary skill in the art by monitoring the amount of amyloid beta peptide that can be detected prior to the contacting step, and which can gradually increase at different times after the contacting step. The time of the experiment can be determined as described in the example in the experimental part.

Thus, in a preferred embodiment of the invention, the parameter determined in step (i) of the method according to the invention is one or more parameters selected from the group consisting of the level of ABETA40 associated with cells present in a biological sample (hereinafter referred to as 3ab40 or CB Α β (1-40)), and the level of ABETA40 associated with cells present in the biological sample (hereinafter referred to as 3ab42 or CB Αβ (1-42)).

The following step of the method according to the invention involves comparing the value of at least one of the parameters (b) or (c) or the value of the parameter calculated by arithmetically combining one or more parameters (a) - (c) with a reference value corresponding to the value of the specified parameters (b ) or (c) or the specified calculated parameter in the reference sample, where (a), (b) and (c) are determined as described above in detail and correspond to the level of free amyloid beta peptide in the biological sample of the indicated individual, respectively (a) the levels of aggregates of the free amyloid peptide in a biological sample taken from the indicated individual and the levels of the indicated amyloid beta peptide associated with the macromolecular components present in the specified biological sample (b), as well as the level of the amyloid beta peptide associated with cells in a biological sample taken from the specified individual (c).

The parameters used in step (ii) of the method according to the invention are any direct parameters, that is, parameters that can be determined directly by the method described above, and which correspond to parameters previously defined as 2ab40, 3ab40, 2ab42 and 3ab42. Alternatively, the calculated parameter can also be obtained using an arithmetic combination of one or more direct parameters. In practice, calculated parameters with a diagnostic value can be obtained by any arithmetic combination of two or more parameters, including addition, subtraction, multiplication, division, and combinations thereof. Specific calculated markers used in the method according to the invention include, but are not limited to, 2ab40 / 2ab42, 3ab40 / 3ab42, 2ab40 / 3ab40, 2ab42 / 3ab42, 1ab40 + 2ab40, 1ab40 + 3ab40, 2ab40 + 3ab40, 1ab40 + 2ab40 + 3ab40 , 1ab42 + 2ab42, 1ab42 + 3ab42, 2ab42 + 3ab42, 1ab42 + 2ab42 + 3ab42, 1ab40 + 2ab40 + 1ab42 + 2ab42, 1ab40 + 3ab40 + 1ab42 + 3ab42, 2ab40 + 3ab40 + 2ab42 + 3ab42, 1ab40 + 2ab40 + 3ab40 + + 2ab42 + 3ab42, (1ab40 + 2ab40) / (1ab42 + 2ab42), (1ab40 + 3ab40) / (1ab42 + 3ab42), (2ab40 + 3ab40) / (2ab42 + 3ab42), (1ab40 + 2ab40 + 3ab40) / ( 1ab42 + 2ab42 + 3ab42), (1ab42 + 2ab42) / (1ab40 + 2ab40), (1ab42 + 3ab42) / (1ab40 + 3ab40), (2ab42 + 3ab42) / (2ab40 + 3ab40), (1ab42 + 2ab42 + 3ab42) / (1ab40 + 2ab40 + 3ab40), 2ab40-1ab40, 2ab42-1ab42 and (2ab40-1ab40) / (2ab42-1ab42). In a preferred embodiment of the invention, the calculated parameter is obtained by adding the parameters 2ab40 and 3ab40 (hereinafter referred to as T40). In another preferred embodiment of the invention, the calculated parameter is obtained by adding the parameters 2ab42 and 3ab42 (hereinafter referred to as T42). In another preferred embodiment of the invention, the calculated parameter is obtained by adding the parameters 2ab40, 3ab40, 2ab42 and 3ab42 (hereinafter referred to as Τ-βΑΡΒ).

As used herein, the term “reference value” means a parameter value that is used for comparison and which has been determined in an individual not suffering from a neurodegenerative disease, or in an individual who has no history of any neurodegenerative disease. Preferably, individuals from whom reference values were obtained for various parameters and calculated parameters are patients who did not have complaints of memory loss, normal behavior was observed, as indicated by neuropsychological tests, and there were no structural changes in MRI.

In particular, reference values were selected that corresponded to sensitivity above 85% and specificity above 75%. In another preferred embodiment of the invention, the reference values are selected so that they correspond to sensitivity above 70% and specificity above 70%. Preferably, the reference values allow prediction with an accuracy of at least 80%.

In step (iii) of the method according to the invention, the diagnosis of a neurodegenerative disease, the determination of the stage preceding the neurodegenerative disease, or the differentiation of the neurodegenerative disease from the stage preceding the neurodegenerative disease, is carried out using a specific series of markers or calculated markers that give sufficiently adequate levels of specificity and sensitivity. Thus, in a specific example, the diagnosis of a neurodegenerative disease is made by comparing the value of a parameter selected from the group consisting of 3ab40 and 2ab42, or the value of the calculated parameter selected from the group consisting of 2ab40 + 3ab40 and 2ab40 + 3ab40 + 2ab42 + 3ab42.

In another specific embodiment of the invention, the detection of a disease stage preceding a neurodegenerative disease is carried out by comparing the value of a parameter selected from the group consisting of 2ab40, 3ab40 and 2ab42, or the value of the calculated parameter selected from the group consisting of 2ab40 + 3ab40, 2ab40 + 3ab40 + 2ab42 + 3ab42, 1ab40 + 2ab40 + 3ab40 + 1ab42 + 2ab42 + 3ab42, 1ab40 + 2ab40 + 3ab40, 1ab42 + 2ab42 + 3ab42, 1ab40 + 1ab42 + 2ab42 + 3ab42, 1ab40 + 2ab40 + 1ab42 + 2ab42 and 1ab40 + 3ab40 + + 3ab42.

In another embodiment of the invention, the differentiation of the neurodegenerative disease from the stage of the disease preceding the specified neurodegenerative disease is carried out by comparing the value of a parameter selected from the group consisting of 3ab40 and 2ab42, or by comparing the value of the calculated parameter selected from the group consisting of 2ab40 + 3ab40, 2ab40 + 3ab40 + 2ab42 + 3ab42 and 1ab40 + 2ab40 + 1ab42 + 2ab42.

Adequate reference values used in various diagnostic methods according to the invention are systematized in table 1.

Table 1
Systematized parameters and threshold values for preferred methods according to the invention Way Parameter Threshold value (pg / ml) Detecting the stage preceding a neurodegenerative disease 2ab40 / DP (Αβ1-40) 63.8 The diagnosis of neurodegenerative disease 3ab40 / CB (Αβ1-40) 71.9 Detecting the stage preceding a neurodegenerative disease 3ab40 / CB (Αβ1-40) 71.1 Differentiation of neurodegenerative 3ab40 / CΒ (Αβ1-40) 211.3 diseases from the stage preceding said neurodegenerative disease The diagnosis of neurodegenerative disease 2ab42 / DP (Αβ1-42) 47.4 Detecting the stage preceding a neurodegenerative disease 2ab42 / DP (Αβ1-42) 50.3 Differentiation of a neurodegenerative disease from a stage preceding said neurodegenerative disease 2ab42 / DP (Αβ1-42) 151.7 The diagnosis of neurodegenerative disease 3ab42 / CB (Αβ1-42) 76.9 Detecting the stage preceding a neurodegenerative disease 3ab42 / CB (Αβ1-42) 58.8 The diagnosis of neurodegenerative disease 2ab40 + 3ab40 / T40 132.7 Detecting the stage preceding a neurodegenerative disease 2ab40 + 3ab40 / T40 132.7 Differentiation of a neurodegenerative disease from a stage preceding said neurodegenerative disease 2ab40 + 3ab40 / T40 550.8 The diagnosis of neurodegenerative disease 2ab42 + 3ab42 / T42 115.8 Detecting the stage preceding a neurodegenerative disease 2ab42 + 3ab42 / T42 103.3 The diagnosis of neurodegenerative disease 2ab40 + 3ab40 + 2ab42 + 3ab42 / T-βARV 235.5 Detecting the stage preceding a neurodegenerative disease 2ab40 + 3ab40 + 2ab42 + 3ab42 / T-βARV 235.5 Differentiation of a neurodegenerative disease from a stage preceding said neurodegenerative disease 2ab40 + 3ab40 + 2ab42 + 3ab42 / T-βARV 778.1 Detecting the stage preceding a neurodegenerative disease 1ab40 + 2ab40 + 3ab40 + 1ab42 + 2ab42 + 3ab42 272.1 Detecting the stage preceding a neurodegenerative disease 1ab40 + 2ab40 + 3ab40 155.8 Detecting the stage preceding a neurodegenerative disease 1ab42 + 2ab42 + 3ab42 124.3 The diagnosis of neurodegenerative disease 1ab40 + 2ab40 + 1ab42 + 2ab42 158.3 Detecting the stage preceding a neurodegenerative disease 1ab40 + 2ab40 + 1ab42 + 2ab42 142.4 Differentiation of a neurodegenerative disease from the stage preceding the specified neurodegenerative disease 1ab40 + 2ab40 + 1ab42 + 2ab42 161.2 Detecting the stage preceding a neurodegenerative disease 1ab40 + 3ab40 + 1ab42 + 3ab42 154.7

After determining the value of the parameter or the calculated parameter, the diagnosis of a neurodegenerative disease, detecting the stage preceding the neurodegenerative disease, or differentiating the neurodegenerative disease from the stage preceding the specified neurodegenerative disease according to the invention, can be achieved if there is a change in the value of the parameter or the value of the calculated parameter compared with a reference value.

The term “change” means a statistically significant increase or decrease in the magnitude of a given parameter compared to a reference value.

As used herein, the term “statistically significant” refers to a statistical probability analysis, which is a non-random combination of two or more results, endpoints, or outcomes of a disease, that is, a certain degree of mathematical certainty that the parameter value associated with a particular group of patients corresponds to a reference size.

A statistically significant change in values can be determined using the p-value. So, for example, when using the p-value, the parameter is identified as a statistically significant change if the p-value is less than 0.1, preferably less than 0.05, more preferably less than 0.01, even more preferably less than 0.005, and most preferably, less than 0.001.

Usually, the value of the considered parameter can be considered “increased” if the indicated value is at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more exceeds the reference value. On the other hand, the value of the parameter can be considered “reduced” if the indicated value is at least 0.9 times, 0.75 times, 0.2 times, 0.1 times, 0.05 times, 0.025 times, 0.02 times, 0.01 times, 0.005 times or less below the reference value. In a particular embodiment of the invention, a change in the value of the parameter or the value of the calculated parameter compared to the reference value is an increase in the specified value.

Any method suitable for determining peptides may be used in the present invention. For example, the concentration of amyloid beta peptide can be determined using one or more methods selected from Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), analysis of surface plasmon resonance, reaction with precipitin, immunodiffusion analysis by diffusion in gel, radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), two-dimensional gel electrophoresis, capillary electrophoresis, mass spectroscopy (MC), matrix laser desorption and / time-of-flight ionizing MS (MALDI-TOF), laser desorption with time-of-flight ionization on an activated surface (SELDI-TOF), high performance liquid chromatography (HPLC), liquid rapid chromatography of proteins (HPLC), multivariate liquid chromatography (MLC), tandem mass -spectrometry (MS / MC), thin-layer chromatography, analysis by expression on a protein chip and laser densitometry.

In a preferred embodiment of the invention, the determination of at least one or more 1ab40, 1ab42, 2ab40, 2ab42, 3ab40 and 3ab42 is carried out by immunological method. As used herein, the term “immunological method”, as used herein, means any method that involves the use of one or more antibodies specific for a target substance to determine the amount / concentration of said target substance, excluding other substances present in the sample. Suitable immunological methods include, but are not limited to, Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance analysis, radioimmunoassay (RIA).

It will be apparent to those skilled in the art that any type of antibody can be used to carry out the immunological detection methods of the invention, provided that the antibody has a specificity sufficient to efficiently differentiate the amyloid beta peptide molecules in the sample from other substances. Antibody molecules suitable for use in the immunological methods of the invention are, but are not limited to:

(i) "intact" antibodies containing an antigen-binding variable region, as well as a constant domain of the light chain (CL) and constant domains of the heavy chain CHl, CH2 and CH3,

(ii) “Fab” fragments obtained by hydrolysis of an intact antibody by papain and containing one antigen-binding site and a region of CL and CH1,

(iii) "F (ab ') ₂ " fragments obtained by hydrolysis of an intact antibody with pepsin and containing two antigen-binding sites,

(iv) "Fab" fragments containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and only one antigen binding site. Fab'fragments differ from Fab fragments in that several residues are attached to them at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines originating from the hinge region of the antibody.

(v) “Fv,” which is a minimal antibody fragment containing a full-sized antigen recognition site and an antigen-binding site. This region consists of a dimer consisting of one variable domain of the heavy chain and one variable domain of the light chain, strongly connected to each other by a non-covalent bond. This configuration is three hypervariable regions (CDRs) of each variable domain that make up a specific antigen-binding site on the surface of the VH-VL dimer. In total, six hypervariable regions confer antigen binding specificity to the antibody. However, even one variable domain (or half of the Fv, containing only three hypervariable regions specific for the antigen) has the ability to recognize the antigen and bind to the antigen, albeit with less affinity than the full-sized binding site.

(vi) Single chain Fv or “scFv” fragments of an antibody comprising the VL and VH domains of an antibody, wherein said domains are present on the same polypeptide chain. Preferably, the VL and VH regions are connected by a polypeptide linker, which allows the scFv fragment to form the desired structure suitable for binding to the antigen.

(vii) “Dibodies” comprising a heavy chain variable domain (VH) coupled to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) via a peptide linker that is too short to form pairs between two domains on the same chain. This ensures pairing with complementary domains of another chain and stimulates the assembly of a dimeric molecule with two functional antigen-binding sites.

(viii) “Bispecific antibodies” (BAb), which are single chain divalent antibodies (or immunotherapeutically effective fragments thereof) having two antigen-binding sites with different specificity. These two antigen-binding sites can be linked to each other by a chemical method or genetic engineering methods known to those skilled in the art.

All of these antibody fragments can also be modified by standard methods known to those skilled in the art, for example, using amino acid (s) deletion (s), insertion (s), substitution (s), addition (s) and / or recombination (and / or any (s) other (s) modification (s) (for example, known post-translational and chemical modifications, such as glycosylation and phosphorylation), either individually or in combination, Methods for introducing such modifications into a DNA sequence encoding the corresponding amino acid sequence of an immuno globulin chain, well known to those skilled in the art; see, for example, Sambrook et al; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2 ^nd edition 1989 and 3 ^rd edition 2001.

Antibodies can be either polyclonal or monoclonal antibodies. To obtain polyclonal antibodies, various hosts, including goats, rabbits, rats, mice, camels, one-humped camels, llamas, humans, birds, etc., can be immunized by injecting a peptide corresponding to a fragment of Αβ40 or Αβ42 having immunogenic properties. Depending on the type of host, various adjuvants may be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels, such as aluminum hydroxide, and surfactants, such as lysolecithin, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among the adjuvants used for administration to humans, BCG (Calmett-Guerin Bacillus) and Corynebacterium parvum are particularly preferred. If the antigen is a peptide, then it can be used for conjugation with a protein that is immunogenic for immunized species. So, for example, the antigen can be conjugated with cochlear lymph hemocyanin (KLH), Blue Carrier (hemocyanin isolated from Concholepas concholepas ), bovine thyroglobulin or a soybean trypsin inhibitor through a bifunctional or derivatizing agent, for example, maleimidobenzoin-aminosulfide, N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic acid anhydride or SOCl ₂ .

Standard methods can be used to produce monoclonal antibodies. For example, monoclonal antibodies can be obtained by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), in accordance with the procedure described in detail in sections 11.4-11.11 of Ausubel, FM et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc; ring-bound edition, 2003). Alternatively, monoclonal antibodies can be isolated by recombinant DNA methods from phage libraries of antibodies obtained by the methods described in McCafferty et al., Nature, 348: 552-554 (1990). Clacksoii et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol, 222: 581-597 (1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Other publications describe the production of high-affinity (in the nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination, used as a strategy for the construction of very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21: 2265-2266 (1993)). Thus, these methods are a real alternative to traditional hybridoma technology for the production of monoclonal antibodies.

Polyclonal antibodies can be used directly as antiserum obtained from immunized hosts after blood sampling and removal of a fibrin clot. Monoclonal antibodies can be used directly as a supernatant of a hybridoma culture or as ascites fluid after implantation of a hybridoma into the abdominal cavity of a suitable host. Alternatively, the immunoglobulin molecules, both polyclonal and monoclonal, can be purified using standard methods, such as affinity purification using peptides derived from amyloid beta peptides, purification in a non-denaturing gel, purification by HPLC or RP-HPLC, purification by size exclusion chromatography, protein A column purification, or any combination of these methods.

Suitable antibodies that can be obtained by immunological methods according to the invention are, but are not limited to:

(i) Antibodies that recognize a region starting from the N-terminal region of amyloid beta peptides, such as antibodies specific for epitopes localized at amino acid positions 1-16, 1-17, 13-28, 15-24, 1- 5 and 1-11 peptides Αβ40 or Αβ42; and antibodies produced against a peptide corresponding to the C-terminal region of the ββ42 peptide that specifically bind to ββ42 and do not enter into any explicit cross-reaction with ββ40 or ββ43,

(ii) Antibodies produced against a peptide corresponding to the C-terminal region of the Αβ40 peptide that specifically bind to Αβ40 and do not enter into any explicit cross-reaction with Αβ42, Αβ39, Αβ38, Αβ41 or Αβ43,

(iii) Antibodies that simultaneously recognize the C-terminal region of Αβ40 and Αβ42,

(iv) Antibodies that are specific for the junction region of amyloid beta peptides, and which can be used to identify amyloid beta peptides from other APP fragments, and are located at amino acid positions 16 and 17 of the amino acid sequence, usually extending from amino acid residue 13 to amino acid residue 28,

(v) The combination of two or more antibodies referred to in points (i) to (iv).

In a preferred embodiment of the invention, the determination of the amount of amyloid beta peptide is carried out using ELISA.

As used herein, the term “ELISA” means enzyme-linked immunosorbent assay and analysis in which an unknown amount of the target substance (amyloid beta peptide) is fixed on the surface, and then a specific antibody is applied to this surface to bind to the antigen. This antibody binds to the enzyme, and in the final step, a substance is added that the enzyme can turn into a specific detectable signal. Various types of ELISA assays are known to those skilled in the art and can be used in the method of the invention, including direct ELISA, sandwich ELISA, competitive ELISA, and reverse ELISA method and apparatus (ELISA-R m & d).

Direct ELISA analysis is carried out by contacting a test sample containing an amyloid beta peptide with a solid carrier that has been precoated with a concentrated solution of an inert protein or reagent (bovine serum albumin, casein). After absorption of the amyloid beta peptide present in the test sample on the carrier, an antibody specific for the amyloid beta peptide is added under conditions suitable for binding to the amyloid beta peptide. The bound antibody is then detected using a “second” antibody that binds to a detectable label or to a substrate-modifying enzyme. The signal produced by the detectable label or substrate is proportional to the amount of antibody bound to the carrier, which, in turn, directly correlates with the amount of amyloid beta peptide in the sample.

Competitive ELISA analysis involves a first step in which a test sample containing an unknown amount of an amyloid beta peptide is contacted with a “first” antibody as defined above. Antigen-antigen complexes are added to the well coated with antigen. After washing the carrier to remove any non-specifically bound complexes, the amount of the “first” antibody is detected using the “second” antibody bound to the detected molecule. In analyzes of this type, the higher the initial concentration of antigen, the weaker the output signal. An alternative competitive ELISA assay is an ELISA that does not use an antibody bound to an enzyme, but an antigen bound to the enzyme. Labeled antigen competes with the antigen (unlabeled) of the sample for binding sites with the "first" antibody. In analyzes of this type, the concentration of antigen in the sample is inversely proportional to the amount of labeled antigen remaining in the well, and accordingly, the higher the concentration, the weaker the signal.

In the method and device for reverse ELISA (ELISA-R m & d), a new type of solid phase is used, made of an enzyme-linked immunosorbent polystyrene rod with 4-12 protruding pointed ends, and the entire device can be introduced into a test tube containing the collected sample, and subsequent stages (washing, incubation in the form of a conjugate and incubation in a chromogenic solution) can be easily carried out by immersing the pointed ends of the device in microwells in standard microplates pre-filled with reagents and wounding in a sealed form up to their application.

In a preferred embodiment of the invention, the ELISA assay is a sandwich ELISA. The sandwich ELISA involves the application of the “first” antibody specific for the amyloid beta peptide to the carrier, the application of a sample containing the amyloid beta peptide, which leads to the binding of the amyloid beta peptide to the “first” antibody, and the application of the “second” an antibody that is also specific for an amyloid beta peptide, wherein said “second” antibody typically binds to a detectable label or to a substrate-modifying enzyme. The signal generated by the label or converted substrate is proportional to the amount of antigen in the sample.

In the context of the description of the present invention, the "first" antibody is called a "capture antibody", which means that this antibody is used to isolate from a sample molecules of all kinds with which this antibody specifically binds. The type of antibody that can be used as an capture antibody is not particularly limited as long as it contains at least one antigen binding site specific for Αβ40 and / or Αβ42. Thus, any of the above antibodies can be used as capture antibodies.

In the context of the description of the present invention, the "second" antibody is called a "detecting antibody", because this antibody can be used to detect the amount of antigen that is retained by the antibody to capture. Similar to an capture antibody, the type of antibody that can be used as a detecting antibody is virtually unlimited. However, in this regard, it should be noted that the detecting antibody (i) must bind to the region of the antigen that was not sensitized by the capture antibody, and (ii) must contain not only the antigen binding site, but also a detectable label, substrate-modifying enzyme, or additional (s) region (s) that (s) can (can) be specifically detected by a reagent that detects high binding affinity for the specified antibody, which will allow detection of an antibody that binds to the antigen captured by the antibody scrap to capture. Preferably, said additional regions that can specifically bind to said reagent correspond to a constant region of an immunoglobulin molecule.

In a preferred embodiment of the invention, the capture antibody is an antibody specific for the N-terminal region of amyloid beta peptides. In a more preferred embodiment of the invention, said capture antibody is an antibody specific for an epitope localized at amino acid positions 1-16 of Abeta40 or Abeta42. A particularly preferred capture antibody is monoclonal antibody 6E10 described by Kim, K.S. (Neuroscience Res. Comm. 1988, 2: 121-130).

In another preferred embodiment of the invention, the detection antibody is an antibody specific for an epitope located in the C-terminal region of the amyloid beta peptide. As explained above, the type of detecting antibody may be adequately selected by one of ordinary skill in the art, depending on the number of detectable amyloid beta peptides.

An antibody that recognizes the N-terminal region of Αβ40 (as well as Αβ42, since both these peptides have identical N-terminal regions) can be used to detect or determine one Αβ40 antibody for capture, and an antibody that specifically recognizes C- can serve as a detection antibody the terminal region is Αβ40, but does not enter into any cross-reaction with Αβ42. Alternatively, Αβ40 can be specifically detected using a capture antibody that recognizes the C-terminal region of Αβ40, but does not enter into any cross-reaction with Αβ42, and a detecting antibody recognizing the 40β40 region, which is common for Αβ40 and Αβ42 and preferably, the N-terminal region of Αβ42 / Αβ40.

An antibody that recognizes the N-terminal region of Αβ42 (as well as Αβ42, since both these peptides have identical N-terminal regions) can be used to detect or determine one Αβ42 antibody for capture, and an antibody that specifically recognizes C- can serve as a detection antibody the terminal region is Αβ42, but does not enter into any cross-reaction with Αβ40. Alternatively, Αβ42 can be specifically detected using a capture antibody that recognizes the C-terminal region of Αβ42, and a detecting antibody recognizing the Αβ42 region, which is common between Αβ42 and Αβ40.

For the simultaneous detection or determination of Αβ42 and антителβ40, the capture antibody can be an antibody that recognizes the N-terminal region common to Αβ42 and Αβ40, and a combination of at least two antibodies, where the first antibody specifically recognizes the C-terminal region of Αβ42, can serve as a detection antibody but does not enter into any cross-reaction with Αβ40, and the second antibody specifically recognizes the C-terminal region of Αβ40, but does not enter into any cross-reaction with Αβ42. Alternatively, the capture antibody can be an antibody that recognizes an N-terminal region common to Αβ42 and Αβ40, and a detection antibody can be an antibody that recognizes the C-terminal region of Αβ40 and Αβ42. Alternatively, Αβ40 and Αβ42 can be simultaneously detected using an antibody to capture a mixture of at least two antibodies containing the first antibody that specifically recognizes the C-terminal region of Αβ42, but does not enter into any cross-reaction with Αβ40, and a second antibody that specifically recognizes the C-terminal region of Αβ40, but does not enter any cross-reaction with Αβ42, and a detecting antibody that recognizes the N-terminal region common to Αβ42 and Αβ40. Alternatively, Αβ42 and Αβ40 can be simultaneously detected using an antibody that recognizes the C-terminal region of Αβ40 and Αβ42 and a detection antibody that recognizes the N-terminal region common to Αβ42 and Αβ40 as an antibody to capture.

Antibodies specific for Αβ40 and Αβ42, and methods for their preparation, have been described in detail in WO2004024770 and WO2004098631, the contents of which are incorporated herein by reference.

Detecting antibodies and / or capture antibodies can be affinity purified using a polypeptide containing the sequence of the polypeptide used to produce these antibodies.

As mentioned above, a detecting antibody can be directly linked to a detectable label or to a substrate-modifying enzyme. Preferably, a reagent having affinity for a detecting antibody can be used, in which case the reagent is labeled not with a detecting antibody, but with a detectable label or substrate-modifying enzyme. In addition, the antibody-binding reagent can be associated with the first member of the binding pair, and in this case, it is the second member of the binding pair, which may be associated with a detectable label or with a substrate-modifying enzyme.

An antibody-binding reagent may be non-covalently coupled to an antibody of specific type (s), specific class (s) and / or specific subclass (s) and / or fragments of such antibodies. Alternatively, an antibody-binding reagent may be non-covalently coupled to an antibody specific for a particular antigen. In specific embodiments of the invention, the antibody-binding reagent may be non-covalently linked to the Fc region or to the F (ab) region of a detecting antibody. Preferred antibody-binding reagents are protein A, G-protein, protein V, protein L, anti-Fc antibody or antibody-binding fragment and Fc receptor (FcR) or its antibody-binding fragment. Non-limiting examples of antibodies that can non-covalently bind to a detecting antibody are monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, single chain Fv (scFv) single chain antibodies, Fab ′ fragments, F (ab fragments, F (ab ) fragments, Fv (sdFv) linked by a disulfide bond, intratel and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above antibodies. Non-limiting examples of Fc receptors are FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIAα, FcγRIIIB, FcεRIα, FcεRIξ and FcγRIIIAξ.

Suitable binding pairs used for detection are:

- hapten or antigen / antibody, e.g. digoxin and anti-digoxin antibodies

- biotin or biotin analogues (e.g., aminobiotin, iminobiotin or desthiobiotin) / avidin or streptavidin,

- sugar / lectin,

- enzyme and cofactor,

- folic acid / folate,

- double-stranded oligonucleotides that selectively bind to proteins / transcription factors,

- a nucleic acid or nucleic acid analogue / complementary nucleic acid,

a receptor / ligand, for example a steroid hormone receptor / steroid hormone.

It should be noted that the terms “first” and “second” member of the linking pair are relative, and that each of the above members can be considered as the first or second members of the linking pair. In a preferred embodiment of the invention, the first member of the binding pair is biotin or a functionally equivalent variant thereof, and the second member of the binding pair is avidin, streptavidin or a functionally equivalent variant thereof.

In a preferred embodiment of the invention, the second member of the binding pair is streptavidin.

Suitable detecting labels include, but are not limited to, fluorescent molecules (e.g., fluorescein, rhodamine, phycoerythrin, coumarin, oxazine, resorufin, cyanine and their derivatives), luminescent molecules (e.g., Qdot ™ nanoparticles supplied by Quantum Dot Corporation, Palo Alto, CA).

Suitable substrate modifying enzymes are enzymes having the ability to generate a detectable signal, for example, after adding an activator, substrate, amplifier, etc. Enzymes suitable for use in the present invention as detectable labels and their respective substrates are:

- Alkaline phosphatase:

- Chromogenic substrates: substrates based on p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolylphosphate / cyan nitrotetrazolium (BCIP / NPT), fast red / naphthol-AS-TS-phosphate.

- Fluorogenic substrates: 4-methylumbelliferyl phosphate (4-MUP), 2- (5'-chloro-2'-phosphoryloxyphenyl) -6-chloro-4- (3H) -quinazolinone (CPPCQ), 3,6-fluorescein diphosphate (3, 6-FDP), fast blue BB, fast red TR, or diazonium salts of fast red-violet LB.

- Peroxidase:

- Chromogenic substrates based on 2,2-azinobis (3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), o-phenylenediamine (OPT), 3.3 ', 5.5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 3-dimethylaminobenzoic acid (DMAB) and 3-methyl-2-benzothiazoleninghydrazone (MBTH), 3-amino-9-ethylcarbazole tetrahydrochloride (AEC) and 3,3'-diaminobenzidine (DAB).

- Fluorogenic substrates: 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including red Amplex® reagent, bright red Amplex, and reduced dihydroxanthines.

- Glycosidases:

- Chromogenic substrates: o-nitrophenyl-β-D-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and 4-methylumbelliferyl-β-D-galactoside (MUG) for β-D-galactosidase.

- Fluorogenic substrates: resorufin β-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide, 4-methylumbelliferyl-β-D-galactopyranoside, carboxyumbelliferyl-β-D-galactopyranoside-d-fluoro-galactopyranoside-fluoride.

- Oxidoreductases (luciferase):

- Luminescent substrates: luciferin.

Kits according to the invention

The present invention also relates to kits that are suitable for implementing the method according to the invention. Thus, in another aspect, the present invention relates to a kit for determining amyloid beta peptides in a biological sample, wherein said kit contains:

(i) a protein solubilizing agent and

(ii) at least one anti-amyloid beta peptide antibody.

Components (i) and (ii) of this kit are essentially described in the method according to the invention. In a preferred embodiment of the invention, the protein solubilizing agent is a detergent. In an even more preferred embodiment of the invention, said detergent is Tween 20.

In another preferred embodiment of the invention, at least one anti-amyloid beta peptide antibody is selected from the group consisting of:

(i) antibodies against the N-terminal region of the amyloid beta peptide,

(ii) antibodies against the C-terminal region of the amyloid beta peptide, and

(iii) combinations of an antibody against the N-terminal region of the amyloid beta peptide and an antibody against the C-terminal region of the amyloid beta peptide.

In an even more preferred embodiment of the invention, the kit according to the invention comprises an antibody against the N-terminal region of the amyloid beta peptide, which is directed against an epitope located at amino acid positions 1-16 of ABETA40 and ABETA42. In another preferred embodiment of the invention, the kit according to the invention contains an antibody against the C-terminal region of the amyloid beta peptide, which is directed against the peptide and is selected from the group consisting of:

(i) a polyclonal antibody against a peptide corresponding to the C-terminal region of the ABETA42 peptide, wherein said antibody specifically binds to ABETA42 but does not substantially cross-react with ABETA40,

(ii) an anti-peptide polyclonal antibody corresponding to the C-terminal region of the ABETA40 peptide, wherein said antibody specifically binds to ABETA40 but does not substantially cross-react with ABETA42, and

(iii) an antibody that simultaneously recognizes the C-terminal region of ABETA40 and ABETA42 and

(iv) the combination of antibodies according to (i) and (ii).

In a preferred embodiment of the invention, the kit according to the invention also contains a solid carrier. As used herein, the term “carrier” or “surface” means a solid phase, which is a water-insoluble porous or non-porous material that can be made in various forms, such as strips, sticks, particles, including latex particles, magnetic particles, microparticles, spheres, membranes, microtiter plate wells and plastic tubes. In principle, any material can be used as a solid support, provided that it has the ability to bind to a sufficient amount of antibody to capture. Thus, the choice of solid-phase material can be made based on the desired operating parameters of the analysis of this format. Materials suitable for the manufacture of a solid carrier are polymeric materials, and in particular cellulosic materials and materials derived from cellulose, such as fiber paper, for example filter paper, chromatographic paper, fiberglass paper and the like; synthetic or modified natural polymers such as nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl vinyl chloride . which may be used alone or in combination with other materials; glass such as, for example, glass used as biological glass, ceramics, metals, etc. Preferred are non-crosslinked polymers of styrene and carboxylated styrene, or styrene with other active functional groups such as amino, hydroxyl, halogen, and the like. In some cases, copolymers of substituted styrenes and dienes such as butadiene can be used.

The solid carrier and antibody may be supplied separately in the kit, or, alternatively, the carrier may be precoated with an antibody for capture. In this case, the carrier may be treated with a blocking solution after binding to the capture antibody.

Additional components of this kit may include:

- Means for taking a patient sample for analysis,

- Buffers and solutions needed to build standard curves for amyloid beta peptides,

- Buffers and solutions for washing and blocking solid media during analysis,

- Buffers and solutions for applying antibodies to a solid carrier,

- Reagents for detecting a color or fluorogenic signal produced by a detectable label,

- Reagents for stopping the formation of a colored or fluorogenic product produced by a detectable label (for example, 1N H ₂ SO ₄ ),

- A sample containing a mother solution of Αβ40 or Αβ42 peptides or a combination thereof.

In a preferred embodiment of the invention, the kit according to the invention contains two antibodies that can be used in a sandwich ELISA assay. In this case, one of the antibodies, that is, the capture antibody, is immobilized on a solid support. Immobilization can be carried out before binding of the detectable target polypeptide or after binding of the peptide / protein to the capture antibody. In any case, if a solid support is used, it is more convenient to block the excess protein-binding sites on the support before adding a sample containing the target polypeptide to be determined. Preferably, the blocking or neutralization of the peptide-binding sites on the carrier is carried out using the same buffer that was used to wash the complexes after each binding reaction (for example, 50 mM Tris-HCl, pH 8, PBS or TBS, containing, but not necessarily, tween 20) and to which a macromolecular compound (e.g., bovine serum albumin, skimmed milk powder, blocking reagent used in Western blot analysis, casein, lactoalbumin, ovalbumin) was added at concentrations of about 0.05% -10%, pre preferably 1-5%, and more preferably about 3%.

The present invention is described in the following examples, which are illustrative only and should not be construed as limiting the scope of the invention.

Examples

Example 1

Study groups

The study included 40 participants, among them 16 healthy individuals selected as control (HC), 8 patients with mild cognitive impairment, memory loss (MCI), and 16 patients with Alzheimer's disease (AD). All participants were over 65 years old, and each group was half male and female. The demographic characteristics of participants are systematized in table 2.

table 2
Demographic characteristics Characteristics BA MCI HC P ¹ Men / women 8/8 4/4 8/8 - Age (years, average ± src) 78.8 ± 4.7 ^H 77.3 ± 3.6 ^H 70.3 ± 4.1 ^{M, A} 0,0002 Frequency of occurrence of ApoEε4 (%) 34.4 ^H 37.5 ^H 3.1 ^{M, A} 0.002 Education* 1.9 ± 0.9 1.6 ± 0.9 2.4 ± 0.6 0,072 * Education was evaluated by the following scores: 0 = without education; 1 = primary education; 2 = secondary education; 3 = higher education. ^{1 The} Kruskal-Wallis U-test contrasts with the Mann-Whitney U-test. H, M, and A mean significant HC, MCI, and BA, respectively.

Healthy individuals were carefully selected from socially active volunteers living in communes who did not have complaints of memory loss, normal behavior was observed, as indicated by neuropsychological tests, and there were no structural changes, as indicated by quantitative magnetic resonance imaging (MRI).

MCI participants met Mayo's clinical criteria. In addition, for the selection of participants with MCI, it is necessary that CDR = 0.5, and more than 3 points on the Shelten scale (J. Neurol. Neurosurg Psychiatry. 1992; 55: 967-972), which indicates atrophy of the medial portion of the temporal region and a picture of hypometabolism in the parietal and / or temporal region, determined by positron emission tomography, performed using 18-fluorodeoxyglucose (PET-FDG), presumably on AD. Patients with any psychiatric or systemic abnormality that is supposedly not a neurodegenerative disease and that may cause MCI were excluded.

Specific criteria for inclusion in the BA group are a preliminary diagnosis of AD (NINCDS-ADRDA criteria), CDR = 1, MMSE from 16 to 24 points and an assessment of more than 3 points on the Shelten scale for atrophy of the medial portion of the temporal region.

A cognitive diagnostic test for the MCI group and BA group was performed in accordance with the routine clinical assessment of ACE memory loss described in the literature.

Written informed consent was obtained from each participant in the studies (or, in the case of some patients with AD, the consent of their close relatives). Study protocols were reviewed and approved by the Ethical Committee of the Hospital Clinic i Provincial (Barcelona, Spain).

Example 2

Receiving samples

Individuals were bled and Roche CompleteMini pellets (protease inhibitors) of 10 ml each were added. Blood was either immediately centrifuged or stored at 4 ° C and centrifuged on the day of analysis.

Plasma was separated from the cell fraction, and this plasma was collected, separated into 0.5 ml aliquots and placed in Eppendorf tubes. Aliquots can be stored at -80 ° C.

The level of free amyloid in plasma (1ab40 and 1ab42) was determined directly in undiluted clarified plasma obtained as described below. These parameters are further denoted by 1ab40 or UP (undiluted plasma) Αβ (1-40) for Αβ (1-40) and 1ab42 or UP Αβ (1-42) for Aβ (1-42).

Total plasma amyloid, corresponding to free plasma amyloid plus amyloid associated with plasma components, was determined in samples obtained by diluting 150 μl of plasma in 300 μl of dilution buffer (PBS containing 0.5% tween-20). These parameters are referred to below as 2ab40 or DP (diluted plasma) Αβ (1-40) for Αβ (1-40) and 2ab42 or DP Αβ (1-42) for Αβ (1-42).

Cell-bound amyloid beta was determined by diluting the cell plasma fraction (v / v) 1/5 in a dilution sample (PBS containing 0.5% tween-20). These parameters are denoted below by 3ab40 or CB (associated with cells) Αβ (1-40) for Αβ (1-40) and 3ab42 or CB Αβ (1-42) for Αβ (1-42).

Example 3

Sample Processing Conditions

Sample Dilution

To identify the dilution of the sample with the highest optical density, as shown by ELISA analysis, various dilutions and buffer solutions were tested.

Samples were diluted 1/2, 1/3, 1/4, 1/5 and 1/10. It was found that with a dilution of 1/4 and above, the optical density of the sample decreased. The best dilutions are 1/2 and 1/3 dilutions.

Centrifugation sample

Samples can be clarified by centrifugation for 1 minute at 13,000 rpm to remove components in the form of large particles, which can adversely affect immunological detection. The supernatant can be collected and tested immediately or after the dilution described above.

Ultrasound Sample Processing

Samples can be sonicated for 5-10 minutes. Samples treated with ultrasound can be used directly in ELISA assays, or they can be centrifuged for 1 minute at 13000 rpm, and this supernatant can be used directly in ELISA assays. The sonicated samples can also be diluted using appropriate buffers as defined in the previous examples.

Sample pre-clarification

Samples were passed through a 4B-IgGK Sepharose column to remove possible impurities, mainly as described by Fuckumoto et al., (Arch. Neurol, 2003, 60: 958-964). A 4B-IgGK Sepharose column was prepared by reacting CNBr-activated Sepharose with IgGlk in accordance with the following procedure:

- IgGlk (molecular weight (MW) = 150,000) was dissolved in binding buffer (0.1 M NaHCO ₃ , pH 8.3 and NaCl, 0.5 M (1.5 ml for 300 mg agarose)) using 500 μl IgG1k ( 0.6 mg) and 1.5 ml of binding buffer.

- The resin was washed with 1 mm HCl (60 ml for every 300 mg of resin).

- The ligand was mixed with an acid washed resin and incubated overnight at 4 ° C with constant shaking (alternatively, incubation can be carried out for 2 hours at room temperature).

- Then the excess ligand was washed with 5 volumes of binding buffer. Thereafter, unreacted active groups in the resin were blocked using 0.1 M Tris-HCl, pH 8, for 2 hours at room temperature with shaking.

- The resin was washed in three cycles using, alternatively, 0.1 M Tris-HCl, pH 8, + 0.5 M NaCl / 0.1 M sodium acetate, pH 4, + 0.5 M NaCl.

After obtaining Sepharose 4B-IgGK, the following stages of sample processing were performed:

- 300 μl of plasma was contacted with 525 μl of sample buffer and 75 μl of agarose-IgGlk and incubated for 2 hours at 4 ° C with constant stirring.

- Agarose was removed by centrifugation (5 minutes at 1000 rpm).

- 100 μl of the treated sample was added to the wells of microtiter plates on which the capture antibody specific for the N-terminus of amyloid beta peptides was adsorbed and incubated overnight at 4 ° C.

- The amount of amyloid beta peptide present in the clarified sample was determined by ELISA analysis by adding specific anti-ββ40 or anti-ββ42 specific antiserum (1/4000) to the wells for 1 hour at room temperature and with constant shaking.

- Then the samples were incubated with 1/5000-dilution of biotinylated anti-rabbit antibodies for 1 hour at room temperature with constant shaking.

- Then the samples were incubated with 1/4000-dilution of streptavidin associated with peroxidase for 1 hour at room temperature and with shaking.

- Then the reaction was shown with TMB reagent for 30 minutes in the dark.

- The manifestation of the reaction was stopped by the addition of a blocking solution, and the absorbance was read at 450 nm.

Removal of Albumin and IgG

Samples were passed through columns to which albumin and IgG bind. The flow through was analyzed to identify the fraction in which amyloid peptides were present. Albumin was removed using the ProteoExtract Albumin Removal Kit (CALBIOCHEM) according to the manufacturers instructions.

IgG was removed on a Protein A column (Protein A-Sepharose 4 Fast Flow, Amersham Biosciences) according to the manufacturers instructions.

Sample concentration

Samples were concentrated on a microcentrator with a cut-off of 10,000. Amyloid peptides were captured in a passing stream, and high molecular weight proteins were captured in a retentate.

The effects resulting from processing in accordance with various protocols can be systematized as follows:

- Detergents: Tween-20 is a detergent that provides a significant increase in optical density. This effect was not observed if the concentration of Tween-20 increased from 0.05% to 0.1%.

- pH: The pH values at which the best results were achieved were 7 and 8. When determining Aβ40, adequate absorbances were also observed at pH = 9. When determining Aβ42, adequate absorbances were obtained at pH 9 and 5.

- Denaturation conditions: Addition of 0.5 M or 1 M GuHCl or 10% DMSO did not increase the optical density.

- Salts: Higher absorbance values were obtained using NaCl rather than KCl.

- BSA: No differences in optical density levels were observed when the BSA concentration increased from 0.05% to 0.5%.

- Ultrasound treatment: No differences were observed in the case of sonication of the samples before determining the optical density.

- Pre-clarification: No effect was observed in the case of pre-treatment of samples with 4B-IgGk Sepharose.

- Removal of albumin and IgG: Amyloid peptides obviously bind to IgG and not to albumin.

- Concentration: Amyloid peptides are present mainly in retentate.

Example 4

Colorimetric "sandwich" ELISA analysis with signal amplification under the influence of biotin-streptavidin

To increase sensitivity, the signal can be amplified using biotin-streptavidin. The plate was coated with capture antibody (mAb) 6E10 that recognizes amino acids 1-17 in the amyloid peptide Aβ40 and the amyloid peptide Aβ42. The coating was carried out at a concentration of 5 μg / ml in 100 mM carbonate / bicarbonate buffer, pH = 9.6, overnight at 4 ° C. The plate was then blocked with 300 μl of blocking solution (50 mM Tris-HCl, pH 8, 0.2% Tween-20, 0.5% BSA) for 3 hours at room temperature with shaking or for 2 hours at 37 ° C. If necessary, after blocking, the plate can be treated with 100 μl of a 50 mM Tris-HCl solution, pH 8, containing 20 mg / ml trehalose. The plates were allowed to evaporate until a white halo formed, indicating the presence of trehalose. The plates thus treated wrapped in aluminum foil can be stored at 4 ° C and retain their stability for two years.

Samples of standard curves were constructed according to data obtained for 200 pg / ml of a mother solution of peptides Aβ40 and Aβ42 on tablets coated with mAb 6E10 and treated with trehalose. Serial dilutions of 1: 2 in SDB were prepared from these solutions so that the concentrations were 200, 100, 50, 25, 12.5, 6.25 and 3.125 pg / ml. 100 μl of each diluted or undiluted sample was added to the SDB (1/1000000) and incubated overnight at 4 ° C (or for 2 hours at 37 ° C). Samples for determining free plasma amyloid, total plasma amyloid, and cell-bound amyloid in test samples were prepared as described in Example 1 and added to the wells of ELISA plates under conditions similar to the conditions for preparing the standard sample. After that, a detecting antibody (a polyclonal antibody against a peptide corresponding to the C-terminal region of the Αβ42 peptide or a polyclonal antibody against a peptide corresponding to the C-terminal region of the Αβ40 peptide, depending on the specific peptide detected, i.e., Αβ42 or Αβ40) diluted in SDB . 100 μl of antibody was added to each well and incubated for 1 hour at room temperature. After that, 100 μl of biotin-labeled anti-rabbit IgG antibody (SIGMA) was added at a dilution of 1/5000 in SDB and incubated for 1 hour at room temperature with shaking. Then, 100 μl of HRP conjugated streptavidin (from SIGMA) was added to each well at a dilution of 1/4000 in SDB and incubated for 1 hour at room temperature.

The tablet was developed using 100 μl of TMB chromogenic substrate (ZEU Inmunotec). To do this, TMB was added and incubated in the dark for 15-30 minutes. Then, 50 μl of 1N H ₂ SO ₄ used as a blocking solution was added to each well. Optical density at 450 nm was read on a Synergy HT plate reader (BioTek Instruments).

The concentration values (pg / ml) obtained for the samples used to determine the total plasma amyloid (free plasma amyloid together with the amyloid associated with the plasma components) were adjusted to adjust for dilution during the sample preparation stage. Since the dilution was usually 1: 3 (see Example 1), the pg / ml value obtained after reading the optical density was multiplied by 3 to determine the actual concentration of free amyloid aggregates in plasma and amyloid associated with plasma components. Similarly, the pg / ml values obtained from the absorbance values for the samples used to determine the amyloid associated with the cells were also adjusted to adjust for dilution during the sample preparation step. Since the dilution was usually 1: 5 (see Example 1), the pg / ml value obtained after reading the optical density was multiplied by 5 to determine the actual concentration of amyloid bound to the cells.

Between stages, the tablet was washed in an automatic tablet washing device (Elx50 Bio Tek Instruments), programmed for 5 rinses after each time interval. The wash solution contained 50 mM Tris-HCl, pH 8, 0.05% tween-20 and 150 mM NaCl (filtered before use).

Example 5

Fluorescent Sandwich ELISA

The plate was coated with 6E10 antibody in bicarbonate buffer (5 μg / ml) overnight at 4 ° C. The plate was then blocked for 3 hours at room temperature with shaking (300 μl / well). Then, test samples and samples for constructing a standard curve were added to the plates and incubated overnight at 4 ° C. A 1/4000 dilution of a detecting antibody (anti-Aβ40 or anti-Aβ42 antiserum) was added to each well and incubated for 1 hour at room temperature with shaking. After that, serial dilutions of the FITC-conjugated antibody (1/1000, 1/5000, 1/10000 dilutions) were added and incubated for 1 hour at room temperature in the dark. Fluorescence was read at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

Alternatively, the assay was performed using a Quanta-Blu fluorescent substrate (PIERCE), which increases the sensitivity of the ELISA assay. The maximum excitation was observed at 325 nm, and the maximum radiation was observed at 420 nm. Such excitation can be detected in the wavelength range of 315-340 nm, and radiation can be detected in the wavelength range of 370-470 nm. A QuantaBlu working solution was prepared by mixing 9 parts of a QuantaBlu substrate solution with one part of a stable QuantaBlu peroxidase solution (a solution that was stable for 24 hours at room temperature). Incubation can be carried out for a period of time from 1.5 minutes to 90 minutes at room temperature, and reading can be carried out after completion of the reaction or during the reaction without completion (a blue color appears).

The plate was coated with antibody (mAb) 6E10 in bicarbonate buffer (5 μg / ml) overnight at 4 ° C, and then blocked for 3 hours at room temperature with shaking (300 μl / well). Then, various standard curves were obtained using the following concentrations of Αβ42 and Αβ40 peptides:

- 1000, 500, 250, 125, 62.5, 31, 25 and 15.65 pg / ml

- 200, 100, 50, 25, 12.5, 6.25 and 3.125 pg / ml

- 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 pg / ml

- 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.156 pg / ml

- 5, 2.5, 1.25, 0.625, 0.3125, 0.156 and 0.078 pg / ml

- 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 pg / ml.

A detecting antibody (anti-Aβ40 or anti-Aβ42 antiserum) was added (at a dilution of 1/4000) over 1 hour at room temperature with shaking. Then, a HRP-conjugated anti-rabbit IgG antibody was added at a 1/1000 dilution and incubated for 1 hour at room temperature with shaking. To show the color of the reaction mixture, 100 μl of Quanta-Blue working solution was added and incubated for 30 minutes, 60 minutes and 90 minutes at room temperature in the dark. Then fluorescence was read (excitation: 360/40 nm; radiation: 460/40 nm) for 30 minutes, 60 minutes and 90 minutes, without completing the reaction, or after completion of the reaction with a blocking solution.

Example 6

Construction of standard curves according to the data for Αβ40 and Αβ42

In order to construct a standard curve for Αβ40, a lyophilized sample of human Aβ40 was diluted to 10 μg / ml. Samples containing the following concentrations were obtained from the mother liquor (in pg / ml): 25000 pg / ml, 2500 pg / ml, 25 pg / ml, 12.5 pg / ml, 6.25 pg / ml, 3.125 pg / ml, 1.56 pg / ml; 0.78 pg / ml. Samples were obtained in the presence of 1 mM AEBSF protease inhibitor. Then the samples were processed by the method described in the previous examples.

In order to construct a standard curve for Αβ42, a lyophilized human Aβ42 sample was diluted to 10 μg / ml. Samples containing the following concentrations were obtained from the mother liquor (in pg / ml): 25000 pg / ml, 2500 pg / ml, 25 pg / ml, 12.5 pg / ml, 6.25 pg / ml, 3.125 pg / ml, 1.56 pg / ml; 0.78 pg / ml. Samples were obtained in the presence of 1 mM AEBSF protease inhibitor. Then the samples were processed by the method described in the previous examples.

Example 7

Statistical analysis

The reproducibility of the measurement results in different laboratories was evaluated for 16 randomly selected samples by the correlation and concordance coefficient (CCC), which made it possible to evaluate the correspondence between the three readings, i.e., the readings carried out by the authors in their laboratory and the readings of the same sample carried out in two other laboratories by measuring the deviation of 45 ° from the line passing through the origin (line of concordance) ²⁷ . The degree of similarity of samples obtained from the same individual on different days (reproducibility for the same individual) was evaluated by the intragroup correlation coefficient (ICC). The degree of similarity, assessed by these correlation coefficients, was defined as low (0.21-0.40), moderate (0.41-0.60), high (0.61-0.80) and almost absolute (0, 81-1.00). Αβ levels for various diagnostic groups were compared using the Mann-Whitney U test. Spearman analysis was used to evaluate correlations between continuous variables. To reject the null hypothesis, it is necessary that p <0.05. All statistical analyzes, including diagnostic accuracy, were performed using the SAS 9.1 computer program. The graphs shown in FIG. 1-3, built using the statistical computer program PASW.

The following indicators of reliability and reliability of amyloid beta-peptide markers were evaluated in a screening test for patients with AD, MCI and HC.

For each indicated and specific marker, an analysis was performed specifically described below, and the participants were divided into groups: healthy individuals (control) - patients with AD; healthy individuals (control) - patients with MCI and patients with MCI-BA:

Table 3
Classification of patients into groups: patients with AD and healthy individuals (control) (TP: true positive; FP: false positive; TN: true negative; FN: false negative) Actual classification of participants BA ZK Marker classification of participants BA TR FP ZK Fn TN

Table 4
Patient classification: MCI patients and healthy individuals (control) (TP: true positive; FP: false positive; TN: true negative; FN: false negative) Actual classification of participants MCI ZK Marker classification of participants MCI TR FP ZK Fn TN

Table 5
Classification of patients into groups: patients with AD and patients with MCI (TP: true positive; FP: false positive; TN: true negative; FN: false negative) Actual classification of participants BA MCI Marker classification of participants BA TR FP MCI Fn TN

Reliability Criteria :

Sensitivity: means the probability of a correct classification of sick individuals, that is, the probability of obtaining a positive result for a sick individual in this test. Therefore, sensitivity means the ability of a given test to diagnose a given disease.

TP
-―――― Sensitivity = TP + FN

Specificity : means the probability of a correct classification of healthy individuals, that is, the probability of a negative result for a healthy individual. In other words, specificity can be defined as the ability to identify a healthy individual.

TN
-―――― Specificity = TN + FP

Reliability criteria :

Positive prognostic value : means the likelihood of a disease if this test gives a positive result. Therefore, the predicted positive value can be determined based on the number of patients with a positive test result who, ultimately, fell ill with this disease:

TN
-―――― PPV = TN + FP

Negative predictive value : means the likelihood that an individual with a negative test result is actually healthy. This value can be determined by dividing the number of truly negative results by the total number of patients with a negative test result:

TN
-―――― NPV = TN + FN

In addition to concepts such as sensitivity, specificity, and prognostic values, concepts such as likelihood ratio, probability ratio, or odds ratio can also be considered. The last parameter indicates a more likely specific (positive or negative) result in terms of the presence or absence of the disease.

Positive likelihood ratio or positive odds ratio : this ratio is calculated by dividing the probability of a positive result in sick patients by the probability of a positive result in healthy individuals. In short, this ratio is the ratio of the number of truly positive results (sensitivity) to the number of false positive results (1-specificity):

$$PLR=\frac{H\mathrm{at}\mathrm{at}\mathrm{from}t\mathrm{at}\mathrm{and}telbn\mathrm{about}\mathrm{from}tb}{\mathrm{one}-\mathrm{FROM}Pec\mathrm{and}f\mathrm{and}hn\mathrm{about}\mathrm{from}tb}$$

Negative likelihood ratio or negative odds ratio : this ratio is calculated by dividing the probability of a negative result with respect to the presence of the disease by the probability of a negative result with respect to the absence of the disease. Therefore, this ratio is calculated as the ratio of the number of false negative results (1-sensitivity) to the number of truly negative results (specificity):

$$NLR=\frac{\mathrm{one}-H\mathrm{at}\mathrm{at}\mathrm{from}t\mathrm{at}\mathrm{and}telbn\mathrm{about}\mathrm{from}tb}{\mathrm{FROM}Pec\mathrm{and}f\mathrm{and}hn\mathrm{about}\mathrm{from}tb}$$

The ROC curve is presented in a graph, and the area under the ROC curve with 95% CI, the actual classification according to the parameter of (sick / healthy) patients, sensitivity values, specificity values, positive predictive value and negative predictive value with 95% CI are presented in tables .

All statistical tests were conducted with a significance level of 0.05.

Example 8

Reproducibility of Αβ measurements obtained in various laboratories

Sixteen randomly selected samples taken from any participant and their extracts were sent to two independent laboratories to assess the reproducibility of the measurement results obtained in various laboratories. All markers acted similarly in accordance with CCC, which ranged from 0.84 to 0.99 (overall 95% IC ranged from 0.73 to 0.99), which corresponded, in all cases, to the degree of similarity, rated as high similarities or almost absolute similarities (Fig. 1).

The average intanalytical reproducibility of the measurement results for six markers carried out in each laboratory, expressed as the coefficient of variability for wells with three replicates, was 4.31, 5.83, and 8.34 (table 6). The detection limit in these analyzes carried out in three laboratories was 5.31, 3.63 and 1.91 pg / ml for Αβ1-40 and 2.37, 2.04 and 2.45 pg / ml for Αβ1-42.

Example 9

Intra-individual reproducibility of measurement results измеренийβ

The reproducibility of Αβ measurements for four weekly blood samples (BS1-BS4), measured by ICC, ranged from high to almost absolute for all direct markers in the three groups (table 7). On average, for the three diagnostic groups, a higher ICC corresponded to the measurement results of Αβ1-40 and Αβ1-42 in DP (0.93, 95% CI = 0.98-0.80 and 0.93, 95% CI = 0, 98-0.78; respectively).

Example 10

Comparison of diagnostic groups

In accordance with high intraindividual reproducibility of the measurement results, the comparison between groups of participants gave the same picture for four blood samples taken on different days (BS1-BS4), although some p-values for these blood samples (BS) varied very slightly. The following description is based on BS4 measurements.

The first unexpected result was that the concentrations of Αβ1-40 and Αβ1-42, measured in undiluted plasma (UP), were only about 1/3 and 1/4, respectively, of the levels in diluted plasma (DP) for all diagnostic groups (table 8).

Secondly, it was found that the levels of the peptide associated with the cells (CB), directly measured in the cell fraction of the blood sample, were similar to the levels measured in DP. In addition, the levels of Αβ1-40 and Αβ1-42 clearly correlated with the levels measured in the UP, DP, or CB fraction (r = 0.58, 0.71, and 0.71, respectively; p <0.001). Significant correlations were also found between any pairs of six markers directly analyzed in the samples (Αβ1-40 and Aβ1-42 in UP, DP, CB; (table 9)).

In addition, the authors of the present invention found that in patients with MCI and AD the levels of each marker are higher than in healthy individuals of the control group (figure 2, table 8). This increase reached statistical significance in the group of patients with MCI and in the HC group for three Αβ1-40 markers (UP, DP and CB, whose levels increased 2.9, 2.2 and 1.8 times, respectively) and for two Αβ1-42 plasma markers (UP and DP, whose levels increased 3.1 and 1.8 times, respectively). The average level of each marker in the group with AD was almost similar to its average level in the group with MCI, and there were no significant differences between the two groups of patients. Similarly, no statistical differences between the group of patients with AD and the group of healthy control patients were observed, with the exception of the levels of CB-β1-42 (Fig. 2). This lack of significance is most likely due to the wide range of individual measurements within the group with BA (n = 16), which gave the coefficient of variability (CV) 1.5-2.7 times more than in the group with MCI (n = 8 ), and 2.5-5.7 times more than in the HC group (n = 16), for each marker (table 8).

Plasma markers Αβ1-40 and Αβ1-42 (UP and DP), but not CB, significantly correlated with MMSE (Table 9), although their level can be somewhat overestimated, since HC clustering tends to increase punctuation. Indeed, with the exclusion of participants with MMSE ≥ 26, the levels of Αβ1-40 and Αβ1-42 were lower in some patients with severe pathologies (MMSE ≤ 21, n = 5) than in patients with moderate pathology (MMSE 22-25, n = 12), although this difference did not reach statistical significance (data not shown). In addition, three markers Αβ1-42, but not Αβ1-40, showed a significant correlation with the degree of atrophy of the medial part of the temporal region in the right and left hemisphere (table 9).

The authors of the present invention also considered several markers calculated from markers directly analyzed in the samples. In addition to the usual relations Αβ1-42 / Αβ1-40, the sum of DP + CB-Αβ1-40 and the sum of DP + CB-Αβ1-42, which were designated by the authors as common Αβ1-40 (T40) and common Αβ1-42 ( T42), respectively, and the sum of these two quantities, which were designated by the authors as the total βΑΡΒ (Τ-βΑΡΒ). The Αβ1-42 / Αβ1-40 ratios measured for UP, DP, or CB did not reveal significant differences between the groups. However, in the MCI group, the levels of T40, T42, and T-βΑΡΒ increased 2.0, 1.5, and 1.8 times compared with levels in the group of healthy control individuals (p≤0.03) (table 4). A similar average increase in these levels was observed in healthy control individuals (HC) and in patients with AD, but in this case only T42 gave statistical significance (Fig. 2).

Example 11

Diagnostic signs of direct and calculated markers

The direct and calculated parameters mentioned in table 10 were determined by the methods described in the previous examples.

Table 10
List of direct and calculated parameters that were analyzed during the study Direct parameters Αβ40 Αβ42 1ab40 (UP) 1ab42 (UP) 2ab40 (DP) 2ab42 (DP) 3ab40 (CB) 3ab42 (CB) The calculated parameters Sum of markers Αβ40 Sum of markers Αβ42 1ab40 + 2ab40 1ab42 + 2ab42 1ab40 + 3ab40 1ab42 + 3ab42 2ab40 + 3ab40 / T40 2ab42 + 3ab42 / T42 1ab40 + 2ab40 + 3ab40 1ab42 + 2ab42 + 3ab42 The sum of the markers Αβ40 and Αβ42 1ab40 + 2ab40 + 1ab42 + 2ab42 1ab40 + 3ab40 + 1ab42 + 3ab42 2ab40 + 3ab40 + 2ab42 + 3ab42 / T-βAPB 1ab40 + 2ab40 + 3ab40 + 1ab42 + 2ab42 + 3ab42

The predictive value of the above parameters was tested for:

(i) establishing a diagnosis of Alzheimer's disease (by comparing samples taken from healthy individuals with samples taken from patients with Alzheimer's disease, AD / HC),

(ii) establishing a diagnosis of mild cognitive impairment and the stage preceding Alzheimer's disease (by comparing samples taken from healthy individuals with samples taken from patients with mild cognitive impairment, MCI / HC), and

(iii) differentiating mild cognitive impairment from Alzheimer's disease (by comparing samples taken from patients with mild cognitive impairment with samples taken from patients with Alzheimer's disease, MCI / AD).

The diagnostic properties of these analyzes were evaluated by logistic analysis of the results of результатовβ measurements and the clinical diagnosis, considered as the “gold standard”. The results related to sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, and the area under the receiver performance curve (ROC curve) are summarized in Table 11.

Most direct markers and two calculated markers (T40 and Τ-βΑΡΒ) satisfy the criteria considered suitable for differentiating MCI patients from healthy control individuals (ZK), and these markers are of most interest from any practical point of view, especially when more accurate diagnosis. Thus, all direct markers, except for CB Αβ1-42, represented on the ROC curve ≥ 0.8, and four of these markers (DP Αβ1-40, CB Αβ1-40, UP Αβ1-42 and DP Αβ1-42) give accuracy> 80%, which means that 80% of all test results are correct compared to the clinical "gold standard" (Fig. 3). The calculated markers T40 and Τ-βΑΡΒ gave the same accuracy when differentiating the MCI group from the HC group, in contrast to direct markers, and the T42 marker gave a less reliable result (Fig. 3). Due to the high variability of the results of измеренийβ measurements between individuals in the BA group, no critical point was found at which these markers could differentiate BA patients from individuals of the other two groups with acceptable sensitivity and specificity (Fig. 3).

Example 12

Other parameters indicating the highest sensitivity and specificity, and suitable for their use in the present invention, are the parameters shown in table 12.

Methods have been developed that are particularly suitable for differentiating individuals with mild cognitive impairment from healthy individuals, and which use: marker 1ab40 (figure 4), marker 2ab40 (figure 5), marker 3ab40 (Figure 6), marker 1ab42 (figure 7), marker 2ab42 (figure 8), marker 3ab42 (figures 9 and 10), marker 2ab40 + 3ab40 (figure 11), marker 2ab42 + 3ab42 (figure 12), 2ab42 + 3ab42 (figure 13) and 2ab40 + 3ab40 + 2ab42 + 3ab42 (figure 14).

Claims (13)

1. A method for diagnosing a neurodegenerative disease in an individual, comprising the following stages:
(i) determining one or more parameters selected from the group consisting of:
(a) 3ab40 or
(b) the value of the calculated parameter selected from the group consisting of 2ab40 + 3ab40, 2ab40 + 3ab40 + 2ab42 + 3ab42 and 1ab40 + 2ab40 + 1ab42 + 2ab42;
(ii) comparing the value of parameter (a) or the value of the calculated parameter (b) with a reference value corresponding to the value of the specified parameter (a) or the specified calculated parameter (b) in the reference sample,
where the reference value used in stage (ii) to compare the value of the parameter or the value of the calculated parameter is selected from the group consisting of:
(i) 71.9 pg / ml for parameter 3ab40,
(ii) 132.7 pg / ml for parameter 2ab40 + 3ab40,
(iii) 235.5 pg / ml for parameter 2ab40 + 3ab40 + 2ab42 + 3ab42,
(iv) 158.3 pg / ml for parameter 1ab40 + 2ab40 + 1ab42 + 2ab42; and
(iii) diagnosing a neurodegenerative disease, if there is an increase in the value of the parameter or the value of the calculated parameter compared to the reference value,
where the specified biological sample is plasma or blood,
where the neurodegenerative disease is Alzheimer's disease and
Where
1ab40 corresponds to the level of the free ABETA40 peptide in a biological sample of the indicated individual;
1ab42 corresponds to the level of the free ABETA42 peptide in a biological sample of the indicated individual;
2ab40 corresponds to the level of aggregates of the free ABETA40 peptide in the biological sample of the indicated individual and the ABETA40 peptide associated with the components of the specified biological sample, where 2ab40 is determined by calculating the amount of the AVETA40 peptide in the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation ABETA40 peptide from components present in a biological sample;
2ab42 corresponds to the level of aggregates of the free ABETA42 peptide in the biological sample of the indicated individual and the ABETA42 peptide associated with the components of the specified biological sample, where 2ab42 is determined by calculating the amount of the AVETA42 peptide in the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation ABETA42 peptide from components present in a biological sample;
3ab40 corresponds to the level of the ABETA40 peptide associated with cells in a biological sample of the indicated individual, where 3ab40 is determined by calculating the amount of the ABETA40 peptide after contacting the cell fraction of the specified biological sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptides from cells present in the specified sample; and
3ab42 corresponds to the level of the ABETA42 peptide associated with cells in a biological sample of the indicated individual, where 3ab42 is determined by calculating the amount of the ABETA42 peptide after contacting the cell fraction of the specified biological sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptides from cells present in the specified sample. 2. A method for detecting a stage preceding a neurodegenerative disease, wherein said method comprises the steps of:
(i) determining one or more parameters selected from the group consisting of:
(a) 3ab40 or
(b) a calculated parameter selected from the group consisting of 2ab40 + 3ab40, 2ab40 + 3ab40 + 2ab42 + 3ab42 and 1ab40 + 2ab40 + lab42 + 2ab42;
(ii) comparing the value of parameter (a) or the value of the calculated parameter (b) with a reference value corresponding to the value of the specified parameter (a) or the specified calculated parameter (b) in the reference sample,
where the reference value used in stage (ii) to compare the value of the parameter or the value of the calculated parameter is selected from the group consisting of:
(i) 71.1 pg / ml for parameter 3ab40,
(ii) 132.7 pg / ml for parameter 2ab40 + 3ab40,
(iii) 235.5 pg / ml for parameter 2ab40 + 3ab40 + 2ab42 + 3ab42,
(iv) 142.4 pg / ml for parameter 1ab40 + 2ab40 + lab42 + 2ab42;
and
(iii) detecting the stage of the disease preceding the neurodegenerative disease, if there is an increase in the value of the parameter or the value of the calculated parameter compared to the reference value,
where the specified biological sample is plasma or blood,
where the stage preceding the neurodegenerative disease is a mild cognitive impairment, and
Where
1ab40 corresponds to the level of the free ABETA40 peptide in a biological sample of the indicated individual;
1ab42 corresponds to the level of the free ABETA42 peptide in a biological sample of the indicated individual;
2ab40 corresponds to the level of aggregates of the free ABETA40 peptide in the biological sample of the indicated individual and the ABETA40 peptide associated with the components of the specified biological sample, where 2ab40 is determined by calculating the amount of the AVETA40 peptide in the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation ABETA40 peptide from components present in a biological sample;
2ab42 corresponds to the level of aggregates of the free ABETA42 peptide in the biological sample of the indicated individual and the ABETA42 peptide associated with the components of the specified biological sample, where 2ab42 is determined by calculating the amount of the AVETA42 peptide in the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation ABETA42 peptide from components present in a biological sample;
3ab40 corresponds to the level of the ABETA40 peptide associated with cells in a biological sample of the indicated individual, where 3ab40 is determined by calculating the amount of the ABETA40 peptide after contacting the cell fraction of the specified biological sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptides from cells present in the specified sample; and
3ab42 corresponds to the level of the ABETA42 peptide associated with cells in a biological sample of the indicated individual, where 3ab42 is determined by calculating the amount of the ABETA42 peptide after contacting the cell fraction of the specified biological sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptides from cells present in the specified sample. 3. A method for differentiating a neurodegenerative disease from a stage preceding said neurodegenerative disease, wherein said method comprises the steps of
(i) determining one or more parameters selected from the group consisting of:
(a) 3ab40 or
(b) the value of the calculated parameter selected from the group consisting of 2ab40 + 3ab40, 2ab40 + 3ab40 + 2ab42 + 3ab42 and 1ab40 + 2ab40 + 1ab42 + 2ab42;
(ii) comparing the value of parameter (a) or the value of the calculated parameter (b) with a reference value corresponding to the value of the specified parameter (a) or the specified calculated parameter (b) in the reference sample,
where the reference value used in stage (ii) to compare the value of the parameter or the value of the calculated parameter is selected from the group consisting of:
(i) 211.3 pg / ml for parameter 3ab40,
(ii) 550.8 pg / ml for parameter 2ab40 + 3ab40,
(iii) 778.1 pg / ml for parameter 2ab40 + 3ab40 + 2ab42 + 3ab42,
(iv) 161.2 pg / ml for parameter 1ab40 + 2ab40 + lab42 + 2ab42;
and
(iii) differentiating the neurodegenerative disease from the stage preceding the specified neurodegenerative disease, if there is an increase in the value of the parameter or the value of the calculated parameter compared to the reference value,
where the specified biological sample is plasma or blood,
where the neurodegenerative disease is Alzheimer's disease, and the stage preceding the neurodegenerative disease is a mild cognitive impairment, and
Where
1ab40 corresponds to the level of the free ABETA40 peptide in a biological sample of the indicated individual;
1ab42 corresponds to the level of the free ABETA42 peptide in a biological sample of the indicated individual;
2ab40 corresponds to the level of aggregates of the free ABETA40 peptide in the biological sample of the indicated individual and the ABETA40 peptide associated with the components of the specified biological sample, where 2ab40 is determined by calculating the amount of the AVETA40 peptide in the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation ABETA40 peptide from components present in a biological sample;
2ab42 corresponds to the level of aggregates of the free ABETA42 peptide in the biological sample of the indicated individual and the ABETA42 peptide associated with the components of the specified biological sample, where 2ab42 is determined by calculating the amount of the AVETA42 peptide in the specified sample after contacting the specified sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation ABETA42 peptide from components present in a biological sample;
3ab40 corresponds to the level of the ABETA40 peptide associated with cells in a biological sample of the indicated individual, where 3ab40 is determined by calculating the amount of the ABETA40 peptide after contacting the cell fraction of the specified biological sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptides from cells present in the specified sample; and
3ab42 corresponds to the level of the ABETA42 peptide associated with cells in a biological sample of the indicated individual, where 3ab42 is determined by calculating the amount of the ABETA42 peptide after contacting the cell fraction of the specified biological sample with a protein-solubilizing agent under conditions suitable for stimulating dissociation of amyloid beta peptides from cells present in the specified sample. 4. The method according to any one of paragraphs. 1-3, where the specified protein-solubilizing agent is a detergent. 5. The method according to any one of paragraphs. 1-3, where the step of determining at least one or more 1ab40, 1ab42, 2ab40, 2ab42, 3ab40 and 3ab42 is carried out by immunological method. 6. The method of claim 5, wherein said immunological method is an ELISA assay. 7. The method of claim 6, wherein said ELISA assay is a “sandwich” ELISA assay. 8. The method according to claim 7, where the antibody for capture in the "sandwich" ELISA analysis is an antibody against the N-terminal region of the amyloid beta peptide. 9. The method of claim 8, wherein the anti-N-terminal antibody of the amyloid beta peptide is directed against an epitope localized at amino acid positions 1-7 ABETA40 and AVETA42. 10. The method according to any one of paragraphs. 6-9, where the detection antibody in the sandwich ELISA assay is an antibody specific for an epitope located in the C-terminal region of the amyloid beta peptide. 11. The method of claim 10, wherein the antibody specific for the C-terminal region of the amyloid beta peptide is selected from the group consisting of:
(i) a polyclonal antibody against a peptide corresponding to the C-terminal region of the ABETA42 peptide, where the specified antibody specifically binds to ABETA42, but essentially does not enter into any cross-reaction with ABETA40,
(ii) an anti-peptide polyclonal antibody corresponding to the C-terminal region of the ABETA40 peptide, where the antibody specifically binds to ABETA40, but does not substantially cross-react with ABETA42,
(iii) an antibody that recognizes both the C-terminal region of ABETA40 and ABETA42, and
(iv) a combination of antibodies according to (i) and (ii). 12. The method according to any one of paragraphs. 7-9, where the specified detecting antibody is also detected using a reagent having affinity for the specified antibody, which binds to the first member of the binding pair. 13. The method according to p. 12, where the detection is carried out using the second member of the binding pair associated with the detected label.

Images